Calcium-independent calmodulin binding and two-metal-ion catalytic mechanism of anthrax edema factor

Edema factor (EF), a key anthrax exotoxin, has an anthrax protective antigen-binding domain (PABD) and a calmodulin (CaM)-activated adenylyl cyclase domain. Here, we report the crystal structures of CaM-bound EF, revealing the architecture of EF PABD. CaM has N- and C-terminal domains and each domain can bind two calcium ions. Calcium binding induces the conformational change of CaM from closed to open. Structures of the EF-CaM complex show how EF locks the N-terminal domain of CaM into a closed conformation regardless of its calcium-loading state. This represents a mechanism of how CaM effector alters the calcium affinity of CaM and uncouples the conformational change of CaM from calcium loading. Furthermore, structures of EF-CaM complexed with nucleotides show that EF uses two-metal-ion catalysis, a prevalent mechanism in DNA and RNA polymerases. A histidine (H351) further facilitates the catalysis of EF by activating a water to deprotonate 3'OH of ATP. Mammalian adenylyl cyclases share no structural similarity with EF and they also use two-metal-ion catalysis, suggesting the catalytic mechanism-driven convergent evolution of two structurally diverse adenylyl cyclases.     

MD Simulations of Anthrax Edema Factor: Calmodulin Complexes with Mutations in the Edema Factor “Switch A” Region and Docking of 3′-deoxy ATP into the Adenylyl Cyclase Active Site of Wild-Type and Mutant Edema Factor Variants

Abstract

Bacillus anthracis, a spore-forming infectious bacterium, produces an exotoxin, called the edema factor (EF), that functions in part by disrupting internal signalling pathways. When complexed with human host cell calmodulin (CaM), EF becomes an active adenylyl cyclase, producing the internal signal substance cyclic-AMP in an uncontrolled fashion. Recently, the crystal structures for uncomplexed EF and EF:CaM complexes in the presence and absence of a substrate analog (3′-deoxy-ATP), were reported. EF mutational studies have implicated a number of residues important in CaM binding and/or in the generation of the adenylyl cyclase active site, formed by the movements of the EF switch A, B and C regions upon CaM binding. Here we report on the results of molecular dynamics (MD) simulations on two EF:CaM complexes, one containing wild-type EF and the other containing EF in which a cluster of residues in the switch A region (L523, K525, Q526 and V529) have been mutated to alanine. The switch A mutations cause a large increase in the flexibility of the switch C region, the rupture of a number of EF-CaM interactions, an expansion of the car-boxyl-terminal domain of CaM, and a change in the Ca²⁺ ion binding abilities of the CaM that is in complex with EF. The results indicate the importance of the mutated switch A residues in maintaining a compact EF:CaM complex that appears to be a prerequisite for the generation of a fully-functional adenylyl cyclase active site. The effects of mutating key residues (K346, K353, H577, E588, D590 and N639) in the active site region of EF (to alanine) on the ability of EF to bind the 3′-deoxy-ATP substrate analog were also examined. Active-site residue substitutions at positions 583 (N583A) and 577 (H577A) were found to be particularly distruptive for the placement of the adenine ring moiety into the position found in the x-ray crystal structure of the ligand-protein complex.     

Solubilization of Adenylyl Cyclase from Human Myometrium in a αS-Coupled Form

Adenylyl cyclase (AC) was extracted from human myometrium with either non-ionic (Lubrol-PX or Triton X-100) or zwitterionic (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, CHAPS) detergents. The soluble enzyme was stimulated by forskolin, a hydrophobic activator, in the presence of Mg2+ indicating that the catalytic subunit had not been damaged after solubilization. The enzyme was also activated by 5'-guanylyl imidodiphosphate (Gpp(NH)p) showing that the catalytic unit was not separated from stimulatory guanine nucleotide binding protein (Gs) during the extraction. Both activators showed different effects on the stimulatory efficacy and potency of AC activity solobulized with detergents. Gel filtration of Lubrol-PX and CHAPS extracts over a Sepharose CL-2B column partially resolved AC and its complexes. The chromatographic profile for Lubrol-solubilized AC presented a main peak of about 200 kDa whereas CHAPS-solubilized AC showed a dominant peak of about 1100 kDa. The heterodisperse peaks obtained revealed that the catalytic AC subunit was not separated from Gs proteins after gel filtration, and that AC could be associated with other cellular proteins. When Lubrol extract was submitted to anionic-exchange chromatography, the enzyme was purified about 7.5 fold (enzymatic activity of 48.1 pmol/min/mg of protein). The catalytic subunit was co-eluted with both AC-activating proteins Galphas large (52.2 kDa) and Galphas small (48.7 kDa). This is the first demonstration of the stable physical association of AC with both alphas subunits of G proteins in human myometrium.     

腺苷酸环化酶3缺失对小鼠主要嗅觉表皮组织内相关因子及信号通路的影响

主要嗅觉表皮(main olfactory epithelium, MOE)是哺乳动物感知气味分子的主要嗅觉器官。在MOE组织内,大多数嗅觉神经元通过cAMP信号传导通路感知气味信息。作为嗅觉cAMP信号通路的主要成员之一,腺苷酸环化酶3（adenylyl cyclase 3, ac3）基因敲除小鼠嗅觉探测功能丧失。除cAMP信号传导通路外,MOE内AC3相关因子AC2和AC4,以及肌醇1,4,5-三磷酸（inositol 1,4,5-trisphosphate,IP3）信号通路和Sonic Hedgehog（Shh）信号通路均有表达。然而,敲除ac3是否会对ac2和ac4以及IP3和Shh信号通路成员产生影响,尚不清楚。本文以AC3缺失（AC3-/-）及其野生型小鼠（AC3+/+）MOE为材料,采用实时荧光定量PCR（qRT-PCR）和免疫荧光组织化学方法,发现AC3缺失后,MOE内的ac2和ac4,以及IP3信号通路中的IP3受体ip3r1及钙调蛋白calm1和calm2表达水平均明显降低。Shh信号通路中的受体patched(ptch)与smoothened(smo)、以及核转录因子gli1与gli2的表达也受到了影响。总之,AC3基因缺失不但导致小鼠MOE组织中cAMP信号通路受损,同时AC3相关因子,IP3信号通路和Shh信号通路的传导也受到抑制。本文对于阐明AC3基因敲除小鼠嗅觉丧失的原因及其嗅觉探测机制具有重要启示作用。     

The memory enhancing effect of the APP-derived tripeptide Ac-rER is mediated through CRMP2

Photoactivated adenylyl cyclase α (PACα) was originally isolated from the flagellate Euglena gracilis. Following stimulation by blue light it causes a rapid increase in cAMP levels. In the present study, we expressed PACα in cholinergic neurons of Caenorhabditis elegans. Photoactivation led to a rise in swimming frequency, speed of locomotion, and a decrease in the number of backward locomotion episodes. The extent of the light-induced behavioral effects was dependent on the amount of PACα that was expressed. Furthermore, electrophysiological recordings from body wall muscle cells revealed an increase in miniature post-synaptic currents during light stimulation. We conclude that the observed effects were caused by cAMP synthesis because of photoactivation of pre-synaptic PACα which subsequently triggered acetylcholine release at the neuromuscular junction. Our results demonstrate that PACα can be used as an optogenetic tool in C. elegans for straightforward in vivo manipulation of intracellular cAMP levels by light, with good temporal control and high cell specificity. Thus, using PACα allows manipulation of neurotransmitter release and behavior by directly affecting intracellular signaling.     

缺血/再灌注小鼠海马组织cAMP和腺苷环化酶mRNA水平的变化

目的:观测缺血/再灌注小鼠海马组织环磷酸腺苷(cAMP)和腺苷环化酶(AC)mRNA水平,探讨缺血/再灌注发病的分子生物学机制.方法:通过双侧颈总动脉线结、连续3次缺血-再灌注,制作缺血/再灌注动物模型,并设立假手术组;术后29 d、30 d分别测试学习和记忆成绩;应用放射免疫法检测小鼠海马组织cAMP水平,应用原位杂交技术检测ACmRNA水平.结果:与假手术组比较,模型组学习和记忆成绩均降低(P＜0.05),且海马组织cAMP水平也降低(P＜0.05),海马CA1区AC mRNA阳性神经元面密度明显降低(P＜0.05).结论:海马组织cAMP和AC mRNA水平降低可能参与了缺血/再灌注后学习和记忆障碍的分子生物学发病机制.     

成年小鼠前脑NMDA受体参与神经元的动作电位发放

谷氨酸是中枢神经系统主要的快速兴奋性递质。AMPA受体和海人藻酸受体主要参与突触传递,而NMDA受体主要参与突触可塑性。基因操作的方法增强NMDA受体的功能,可以增强动物在正常生理状态下的学习能力,及在组织损伤情况下的反应敏感性。NMDA受体参与生理功能的主要机制是长时程增强（long—term potentiation,LTP）。我们的研究表明,NMDA受体不仅参与刺激前扣带皮层的第五层细胞或刺激白质诱导的突触反应,而且参与在胞体施加去极化跃阶电流诱导的动作电位的发放。钙一钙调蛋白敏感的腺苷酸环化酶1（adenylyl cyclase 1,AC1）和cAMP信号通路可能介导了这些反应。由于扣带皮层神经元在伤害性刺激和痛中发挥重要作用,我们的结果为前脑NMDA受体参与突触传递和动作电位发放,以及与前脑相关的行为,如感受伤害性刺激和痛,提供了一个新的机制。     

Recombinant human serotonin 5A receptors stably expressed in C6 glioma cells couple to multiple signal transduction pathways

Human serotonin 5A (5-HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5-HT5A receptors-expressing cells, accumulation of cAMP by forskolin was inhibited by 5-HT as reported previously. Pertussis toxin-sensitive inhibition of ADP-ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine-sensitive Ca2+ mobilization. On the other hand, 5-HT-induced outward currents were observed using the patch-clamp technique in whole-cell configuration. The 5-HT-induced outward current was observed in 84% of the patched 5-HT5A receptor-expressing cells and was concentration-dependent. The 5-HT-induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5-HT-induced current was significantly attenuated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5-HT-induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5-HT-induced current. IP3 showed a transient increase after application of 5-HT in 5-HT5A receptor-expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP-ribosyl cyclase activity, 5-HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3-sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function.     

Regulation of the adenylyl cyclase signaling system in various types of cultured endothelial cells

We studied the effects of modulators of the adenylyl cyclase pathway on the accumulation of cAMP in endothelial cells isolated from bovine aortas, pig pulmonary arteries, human umbilical veins, and human subcutaneous adipose microvessels. In addition to quantitative differences in the basal levels, cAMP stimulation in different endothelial cell types varied in sensitivity and magnitude in response to both the direct adenylyl cyclase activator forskolin and the β-adrenergic receptor agonist isoproterenol. Furthermore, the ubiquitous phosphodiesterase inhibitor IBMX differentially enhanced both the basal and the stimulated cAMP levels in the various cell types. Histamine caused an elevation of cAMP only in bovine aortic endothelial cells and in human umbilical vein endothelial cells. Treatment of the cells with cholera and pertussis toxins, which uniquely affect G-protein subunits, resulted in divergent elevation of cAMP in the various cells. Thus, in each cell type, a distinct profile of regulation of the cAMP levels was found. Our results suggest that the adenylyl cyclase signaling system in various types of endothelial cells can be differentially regulated at the levels of receptors, G-proteins, adenylyl cyclase, and phosphodiesterase.     

Cytoenzymological analysis of adenylyl cyclase activity and 3':5'-cAMP immunolocalization in chloroplasts of Nicotiana tabacum

In this study a combination of cytoenzymological and immunocytochemical techniques was used in order to demonstrate the presence of cyclic nucleotide metabolism in chloroplasts of higher plants. Catalytic cytochemistry was used to localize adenylyl cyclase activity by means of electron microscope investigation on Nicotiana tabacum cv. Petit Havana leaf fragments. Various immunocytochemical techniques were explored to visualize the presence of the second messenger adenosine 3':5'-cyclic monophosphate. Making use of adenylyl imidodiphosphate as a substrate, the enzyme activity was predominantly located at the intermembrane space of the chloroplast envelope. In order to provide further topographical information, intact, isolated chloroplasts were submitted to the same cytoenzymological procedure and revealed stromal adenylyl cyclase activity. Using high-pressure freezing as a physical fixative to obtain an instantaneous metabolic arrest the cellular vitrified water phase was sublimed under ultra-high vacuum by means of molecular distillation drying, avoiding recrystallization and hence redistribution of small highly diffusible molecules. This sequential combination preserved 3':5'-cAMP epitope retention in chloroplasts as was demonstrated by immunogold labelling. These results further substantiate in a unique way the growing evidence of the presence of an organelle-specific cAMP metabolism in higher plants. Furthermore the data presented support the status of chloroplasts as an excellent model to further investigate cAMP metabolism and to correlate it with a variety of physiological functions.     

Acid-induced unfolding of the amino-terminal domains of the lethal and edema factors of anthrax toxin

The two enzymatic components of anthrax toxin, lethal factor (LF) and edema factor (EF), are transported to the cytosol of mammalian cells by the third component, protective antigen (PA). A heptameric form of PA binds LF and/or EF and, under the acidic conditions encountered in endosomes, generates a membrane-spanning pore that is thought to serve as a passageway for these enzymes to enter the cytosol. The pore contains a 14-stranded transmembrane beta-barrel that is too narrow to accommodate a fully folded protein, necessitating that LF and EF unfold, at least partly, in order to pass. Here, we describe the pH-dependence of the unfolding of LF(N) and EF(N), the 30kDa N-terminal PA-binding domains, and minimal translocatable units, of LF and EF. Equilibrium chemical denaturation studies using fluorescence and circular dichroism spectroscopy show that each protein unfolds via a four-state mechanism: N<-->I<-->J<-->U. The acid-induced N-->I transition occurs within the pH range of the endosome (pH 5-6). The I state predominates at lower pH values, and the J and U states are populated significantly only in the presence of denaturant. The I state is compact and has characteristics of a molten globule, as shown by its retention of significant secondary structure and its ability to bind an apolar fluorophore. The N-->I transition leads to an overall 60% increase in buried surface area exposure. The J state is expanded significantly and has diminished secondary structure content. We analyze the different protonation states of LF(N) and EF(N) in terms of a linked equilibrium proton binding model and discuss the implications of our findings for the mechanism of acidic pH-induced translocation of anthrax toxin. Finally, analysis of the structure of the transmembrane beta-barrel of PA shows that it can accommodate alpha-helix, and we suggest that the steric constraints and composition of the lumen may promote alpha-helix formation.     

miR‐142‐3p restricts cAMP production in CD4+CD25− T cells and CD4+CD25+ TREG cells by targeting AC9 mRNA

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates diverse cellular functions. It has been found that CD4⁺CD25⁺ regulatory T (T_REG) cells exert their suppressor function by transferring cAMP to responder T cells. Here, we show that miR-142-3p regulates the production of cAMP by targeting adenylyl cyclase (AC) 9 messenger RNA in CD4⁺CD25⁻ T cells and CD4⁺CD25⁺ T_REG cells. miR-142-3p limits the level of cAMP in CD4⁺CD25⁻ T cells by inhibiting AC9 production, whereas forkhead box P3 (FOXP3) downregulates miR-142-3p to keep the AC9/cAMP pathway active in CD4⁺CD25⁺ T_REG cells. These findings reveal a new molecular mechanism through which CD4⁺CD25⁺ T_REG cells contain a high level of cAMP for their suppressor function, and also suggest that the microRNA controlling AC expression might restrict the final level of cAMP in various types of cells.