Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand

In a variety of cells, the Ca2+ signalling process is mediated by the endoplasmic-reticulum-membrane-associated Ca2+ release channel, inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Being ubiquitous and present in organisms ranging from humans to Caenorhabditis elegans, InsP3R has a vital role in the control of cellular and physiological processes as diverse as cell division, cell proliferation, apoptosis, fertilization, development, behaviour, memory and learning. Mouse type I InsP3R (InsP3R1), found in high abundance in cerebellar Purkinje cells, is a polypeptide with three major functionally distinct regions: the amino-terminal InsP3-binding region, the central modulatory region and the carboxy-terminal channel region. Here we present a 2.2-A crystal structure of the InsP3-binding core of mouse InsP3R1 in complex with InsP3. The asymmetric, boomerang-like structure consists of an N-terminal beta-trefoil domain and a C-terminal alpha-helical domain containing an 'armadillo repeat'-like fold. The cleft formed by the two domains exposes a cluster of arginine and lysine residues that coordinate the three phosphoryl groups of InsP3. Putative Ca2+-binding sites are identified in two separate locations within the InsP3-binding core.     

The amino-terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule

Many physiological events require transient increases in cytosolic Ca(2+) concentrations. Ryanodine receptors (RyRs) are ion channels that govern the release of Ca(2+) from the endoplasmic and sarcoplasmic reticulum. Mutations in RyRs can lead to severe genetic conditions that affect both cardiac and skeletal muscle, but locating the mutated residues in the full-length channel structure has been difficult. Here we show the 2.5?? resolution crystal structure of a region spanning three domains of RyR type 1 (RyR1), encompassing amino acid residues 1-559. The domains interact with each other through a predominantly hydrophilic interface. Docking in RyR1 electron microscopy maps unambiguously places the domains in the cytoplasmic portion of the channel, forming a 240-kDa cytoplasmic vestibule around the four-fold symmetry axis. We pinpoint the exact locations of more than 50 disease-associated mutations in full-length RyR1 and RyR2. The mutations can be classified into three groups: those that destabilize the interfaces between the three amino-terminal domains, disturb the folding of individual domains or affect one of six interfaces with other parts of the receptor. We propose a model whereby the opening of a RyR coincides with allosterically coupled motions within the N-terminal domains. This process can be affected by mutations that target various interfaces within and across subunits. The crystal structure provides a framework to understand the many disease-associated mutations in RyRs that have been studied using functional methods, and will be useful for developing new strategies to modulate RyR function in disease states.     

Inositol-1,4,5-trisphosphate receptor regulates hepatic gluconeogenesis in fasting and diabetes

In the fasted state, increases in circulating glucagon promote hepatic glucose production through induction of the gluconeogenic program. Triggering of the cyclic AMP pathway increases gluconeogenic gene expression via the de-phosphorylation of the CREB co-activator CRTC2 (ref. 1). Glucagon promotes CRTC2 dephosphorylation in part through the protein kinase A (PKA)-mediated inhibition of the CRTC2 kinase SIK2. A number of Ser/Thr phosphatases seem to be capable of dephosphorylating CRTC2 (refs 2, 3), but the mechanisms by which hormonal cues regulate these enzymes remain unclear. Here we show in mice that glucagon stimulates CRTC2 dephosphorylation in hepatocytes by mobilizing intracellular calcium stores and activating the calcium/calmodulin-dependent Ser/Thr-phosphatase calcineurin (also known as PP3CA). Glucagon increased cytosolic calcium concentration through the PKA-mediated phosphorylation of inositol-1,4,5-trisphosphate receptors (InsP(3)Rs), which associate with CRTC2. After their activation, InsP(3)Rs enhanced gluconeogenic gene expression by promoting the calcineurin-mediated dephosphorylation of CRTC2. During feeding, increases in insulin signalling reduced CRTC2 activity via the AKT-mediated inactivation of InsP(3)Rs. InsP(3)R activity was increased in diabetes, leading to upregulation of the gluconeogenic program. As hepatic downregulation of InsP(3)Rs and calcineurin improved circulating glucose levels in insulin resistance, these results demonstrate how interactions between cAMP and calcium pathways at the level of the InsP(3)R modulate hepatic glucose production under fasting conditions and in diabetes.     

Kinetics of smooth and skeletal muscle activation by laser pulse photolysis of caged inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) can stimulate skinned smooth and skeletal muscle to contract by initiating Ca2+ release from the sarcoplasmic reticulum. Whether this process is an integral component of the in vivo muscle activation mechanism was tested by releasing InsP3 rapidly within skinned muscle fibers of rabbit main pulmonary artery and frog semitendinosus. InsP3 was liberated on laser pulse photolysis of a photolabile but biologically inactive precursor of InsP3 termed caged InsP3. Caged InsP3 is a mixture of compounds in which InsP3 is esterified with 1(2-nitrophenyl)diazoethane (probably at the P4- or P5-position). Photochemical release of InsP3 induced a full contraction in both muscles at physiological free Mg2+ concentrations, but only in the smooth muscle were the InsP3 concentration (0.5 microM) and the activation rate compatible with the in vivo physiological response. Endogenous InsP3-specific phosphatase activity was present in smooth muscle and had about 35-fold greater activity than that in the skeletal-muscle preparation. Caged InsP3 was not susceptible to phosphatases in either preparation.     

多巴胺D3R蛋白同源模建及其应用

以分辨率为2.2的牛视紫红质蛋白的晶体结构为模板,采用同源模建方法,建立D3R模蛋白。对接D3R模蛋白与刺桐属配体分子,在对接的D3R蛋白的结合腔中选定一个以药物分子为质心,以半径为6的空间范围,计算此空间范围内的所有氨基酸残基与配体分子的作用能量,即残基/配体的结合能或排斥能,据此得到配体分子与受体蛋白的活性结合位点。     

Expression and inhibitory activity analysis of a 25-kD Bowman-Birk protease inhibitor in rice

Bowman-Birk protease inhibitors (BBI), belonging to serine protease inhibitors, are well-studied storage and defense proteins widely existing in both dicoty- ledonous and monocotyledonous plants[1―3]. In dicoty- ledonous plants, the BBIs are about 8-kD p…     

La0.7Ca0.3MnO3材料中取向畴结构的电镜研究

利用透射电子显微镜(TEM)及高分辨电子显微镜(HREM)研究了庞磁电阻材料La0.7Ca0.3MnO3(LCMO)中的取向畴结构.电子衍射结果表明,块体LCMO材料中只存在正交相(Pnma),其衍射指数满足消光条件:0kl,k+l=2n+1;hk0,h=2n+1;h00,h=2n+1;0k0,k=2n+1;00l,l=2n+1.在晶粒中可观察到两种典型的取向畴形貌,分别为无定形和楔形.高分辨观察结果还发现,LCMO畴界均为不平直界面,由一些低能量高指数界面组成.     

InsP4 facilitates store-operated calcium influx by inhibition of InsP3 5-phosphatase

Receptor-mediated generation of inositol 1,4,5-trisphosphate (InsP3) initiates Ca2+ release from intracellular stores and the subsequent activation of store-operated calcium influx. InsP3 is metabolized within seconds by 5-phosphatase and 3-kinase, yielding Ins(1,4)P2 and inositol 1,3,4,5-tetrakisphosphate (InsP4), respectively. Some studies have suggested that InsP4 controls Ca2+ influx in combination with InsP3 (refs 3 and 4), but another study did not find the same result. Some of the apparent conflicts between these previous studies have been resolved; however, the physiological function of InsP4 remains elusive. Here we have investigated the function of InsP4 in Ca2+ influx in the mast cell line RBL-2H3, and we show that InsP4 inhibits InsP3 metabolism through InsP3 5-phosphatase, thereby facilitating the activation of the store-operated Ca2+ current I(CRAC) (ref. 9). Physiologically, this mechanism opens a discriminatory time window for coincidence detection that enables selective facilitation of Ca2+ influx by appropriately timed low-level receptor stimulation. At higher concentrations, InsP4 acts as an inhibitor of InsP3 receptors, enabling InsP4 to act as a potent bi-modal regulator of cellular sensitivity to InsP3, which provides both facilitatory and inhibitory feedback on Ca2+ signalling.     

Irreversible inhibition of Ca2+ release in saponin-treated macrophages by the photoaffinity derivative of inositol-1, 4, 5-trisphosphate

D-myo-inositol-1,4,5-trisphosphate (InsP3) is a putative intracellular second messenger for the mobilization of Ca2+ from intracellular stores, in particular, the endoplasmic reticulum. Specific binding sites on the endoplasmic reticulum may participate in the InsP3-induced release of Ca2+ from the Ca2+ pool. To examine the specific binding sites on the endoplasmic reticulum, we synthesized an arylazide derivative of InsP3 for photoaffinity labelling; InsP3 coupled to p-azidobenzoic acid (InsP3-pAB) using N,N'-carbonyldiimidazole (CDI) was obtained at a 9-11% yield. Here, we report that InsP3-pAB, but not an arylazide derivative of inositol-1,4-bisphophate (Ins(1,4)P2), causes the irreversible inhibition of InsP3-induced release of Ca2+ in saponin-permeabilized photo-irradiated macrophages. The irreversible inhibition by InsP3-pAB after photo-irradiation was prevented by a 10-fold excess of unmodified InsP3.     

A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP6 in mRNA export

Superfamily 1 and superfamily 2 RNA helicases are ubiquitous messenger-RNA-protein complex (mRNP) remodelling enzymes that have critical roles in all aspects of RNA metabolism. The superfamily 2 DEAD-box ATPase Dbp5 (human DDX19) functions in mRNA export and is thought to remodel mRNPs at the nuclear pore complex (NPC). Dbp5 is localized to the NPC via an interaction with Nup159 (NUP214 in vertebrates) and is locally activated there by Gle1 together with the small-molecule inositol hexakisphosphate (InsP(6)). Local activation of Dbp5 at the NPC by Gle1 is essential for mRNA export in vivo; however, the mechanistic role of Dbp5 in mRNP export is poorly understood and it is not known how Gle1(InsP6) and Nup159 regulate the activity of Dbp5. Here we report, from yeast, structures of Dbp5 in complex with Gle1(InsP6), Nup159/Gle1(InsP6) and RNA. These structures reveal that InsP(6) functions as a small-molecule tether for the Gle1-Dbp5 interaction. Surprisingly, the Gle1(InsP6)-Dbp5 complex is structurally similar to another DEAD-box ATPase complex essential for translation initiation, eIF4G-eIF4A, and we demonstrate that Gle1(InsP6) and eIF4G both activate their DEAD-box partner by stimulating RNA release. Furthermore, Gle1(InsP6) relieves Dbp5 autoregulation and cooperates with Nup159 in stabilizing an open Dbp5 intermediate that precludes RNA binding. These findings explain how Gle1(InsP6), Nup159 and Dbp5 collaborate in mRNA export and provide a general mechanism for DEAD-box ATPase regulation by Gle1/eIF4G-like activators.     

The B-cell binding site on human immunoglobulin E

Immunoglobulin E comprises the main immunoglobulin class associated with allergy. Its multifarious activities are mediated by two types of Fc receptors found on different cell populations, Fc epsilon R1 on mast cells and basophils, and Fc epsilon R2 on inflammatory cells (monocytes, eosinophils and platelets) and B lymphocytes. Recombinant epsilon-chain fragments synthesized in Escherichia coli have provided the means of mapping the receptor-binding sites on human IgE, and blocking IgE-receptor interactions. We have previously shown that the Fc epsilon R1 binding site is contained within a sequence (Gln 301-Arg 376) spanning the C epsilon 2 and C epsilon 3 domains. Here we show that Fc epsilon R2 can recognize a motif in the C epsilon 3 domain that is formed on dimerization of one or both of the flanking (C epsilon 2 and C epsilon 4) domains. Glycosylation of IgE is not required for the activity of either receptor.     

菠菜转录因子MYB基因家族的鉴定及表达分析

利用生物信息学手段鉴定了76个具有典型R结构的菠菜转录因子（Spinacia oleracea） MYB,其中包括72条R2R3-MYB基因（2R-MYB）和4条R1R2R3-MYB基因（3R-MYB）。通过生物信息学对菠菜MYB转录因子家族成员的理化性质、染色体定位、结构域序列保守性和系统进化关系进行了分析,结果表明：菠菜MYB家族有32个基因位于染色体正链,另外44个基因位于染色体反链;MYB的DNA结合域中的保守域主要位于两个R重复序列的第二和第三螺旋之间,结合域中每个R重复的第一和第二色氨酸之间的氨基酸序列相对不保守;根据菠菜、拟南芥及甜菜的MYB家族系统进化关系可以推测,菠菜MYB家族中56个成员可以按功能划分为4类,在菠菜的生长发育过程中可能起着重要的调节作用,其余成员中有7个MYB基因可能参与菠菜响应氮素浓度的氮素利用及生长发育进程。     

Quantal calcium release by purified reconstituted inositol 1,4,5-trisphosphate receptors.

Release of intracellular Ca2+ by inositol 1,4,5-trisphosphate (InsP3) occurs through specific receptor proteins which are ligand-activated Ca2+ channels. Changes in intracellular Ca2+ regulate many cellular functions. This Ca2+ release is a discontinuous quantal process in which successive increments of InsP3 transiently release precise amounts of Ca2+ (refs 4-6). Possible explanations of quantal Ca2+ release have included rapid degradation of InsP3, reciprocity of Ca2+ release and sequestration, desensitization of InsP3 receptors, or actions of InsP3 on discrete compartments of Ca2+ with variable sensitivity to InsP3 (ref. 4). We successfully reconstituted InsP3-induced Ca2+ flux in vesicles containing only purified InsP3 receptor protein. The reconstituted vesicles retain the regulatory features of the InsP3 receptor, including phosphorylation sites and modulation of Ca2+ release by adenine nucleotides. Using these reconstituted vesicles, we show here that quantal flux of Ca2+ elicited by InsP3 is a fundamental property of its receptor.     

An inositol tetrakisphosphate-containing phospholipid in activated neutrophils

Inositol (1,4,5)triphosphate (InsP3) and tetrakisphosphate (InsP4) have been observed in a variety of cell types and have been proposed to play roles in the receptor-mediated rise in intracellular Ca2+ (refs 2, 3). Recently, they have been shown to act synergistically in the activation of a Ca2+-dependent K+ channel in lacrimal acinar cells. InsP3 is the product of phospholipase C (PLC) action on phosphatidylinositol 4,5-bisphosphate (PtdInsP2) whereas InsP4 is believed to arise from phosphorylation of InsP3 by a cytosolic kinase. Although sought as a source for InsP4, PtdInsP3 has not been identified in any specific cell type. There were early reports of InsP4-containing phospholipids in crude extract from bovine brain, but this finding was later withdrawn. Recently, however, a membrane-bound enzyme (Type 1 PI kinase) which adds phosphate onto the 3 position of inositol phospholipids has been identified and the phosphatidylinositol-3-phosphate (PtdIns(3)P) product characterized. This suggests that several forms of phosphoinositides may exist and could be precursors for some of the variety of soluble inositol phosphate products which have been reported in recent years. Here we report the appearance of another novel phosphoinositide containing four phosphates, phosphatidylinositol trisphosphate (PtdInsP3) which we find only in activated but not in unstimulated neutrophils from human donors.     

Krull整环中w-理想的直和

主要给出关于Dedekind整环的两个经典结果在Krull整环上的体现.利用w-算子理论,证明了若R是Krull整环,A、B是R的非零理想,则AwBw■R(AB)w·进一步地,结合模的外幂的相关结果,证明了若R是Krull整环,I1,…,Im、J1,…,Jn是R的非零理想,则(I1)w…(Im)w■(J1)w…(Jn)w当且仅当m=n,且存在x∈K-0,使得(I1…In)w=x(J1…Jn)w.     

Reversible inactivation of K+ channels of Vicia stomatal guard cells following the photolysis of caged inositol 1,4,5-trisphosphate

RECENT investigations suggest that cytoplasmic D-myo-inositol 1,4,5-trisphosphate (InsP3) functions as a second messenger in plants, as in animals, coupling environmental and other stimuli to intracellular Ca2+ release. Cytoplasmic levels of InsP3 and the turnover of several probable precursors in plants are affected by physiological stimuli--including light, osmotic stress and the phytohormone indoleacetic acid--and InsP3 activates Ca2+ channels and Ca2+ flux across plant vacuolar and microsomal membranes. Complementary data also link changes in cytoplasmic free Ca2+ to several physiological responses, notably in guard cells which regulate gas exchange through the stomatal pores of higher plant leaves. Recent evidence indicates that guard cell K+ channels and, hence, K+ flux for stomatal movements may be controlled by cytoplasmic Ca2+. So far, however, direct evidence of a role for InsP3 in signalling in plants has remained elusive. Here we report that InsP3 released from an inactive, photolabile precursor, the P5-1-(2-nitrophenyl)ethyl ester of InsP3 (caged InsP3) reversibly inactivates K+ channels thought to mediate K+ uptake by guard cells from Vicia faba L. while simultaneously activating an apparently time-independent, inward current to depolarize the membrane potential and promote K+ efflux through a second class of K+ channels. The data are consistent with a transient rise in cytoplasmic free Ca2+ and demonstrate that intact guard cells are competent to use InsP3 in signal cascades controlling ion flux through K+ channels.     

GTP-activated communication between distinct inositol 1,4,5-trisphosphate-sensitive and -insensitive calcium pools

Inositol 1,4,5-trisphosphate (InsP3) is an established mediator of intracellular Ca2+ signals but little is known of the nature and organization of Ca2+ regulatory organelles responsive to InsP3. Here we derive new information from the study of Ca2+ movements induced both by InsP3 and a specific GTP-activated Ca2+ translocation process. The latter mechanism is clearly distinct from that activated by InsP3 and may involve the translocation of Ca2+ between compartments without its release into the cytosol. This idea is supported by the fact that GTP activates Ca2+ movement into the InsP3-releasable pool. In the light of this evidence we postulated that there are two intracellular Ca2+ pools distinguishable by InsP3-sensitivity and oxalate-permeability, and that movement between them is activated by GTP. We report here direct evidence for the existence and separation of two distinct Ca2+-pumping compartments with properties coinciding with those predicted. Of these, the InsP3-sensitive Ca2+ pool is identified within a purified rough endoplasmic reticulum fraction, an observation consistent with recent InsP3 receptor-localization studies. Ca2+ translocation between pools may reflect function of a class of small GTP-binding proteins known to mediate interorganelle transfer in eukaryotic cells.     

Occurrence and extracellular actions of inositol pentakis- and hexakisphosphate in mammalian brain

Although inositol 1,3,4,5,6-pentakisphosphate (InsP5) and hexakisphosphate (InsP6) have been recognized for some time as naturally-occurring metabolites of inositol, their occurrence in mammalian cell types, including one of neural origin, has only recently been documented. This is of interest because of the recognized second messenger role of inositol 1,4,5-trisphosphate (InsP3) in intracellular signalling; coupling surface stimuli to cytoplasmic calcium discharge. The metabolism, existence in normal mature tissues, and possible functional roles of these inositol polyphosphates are unknown. Here we report evidence that InsP5 and InsP6 are synthesized in intact brain after labelling with [3H]inositol in vivo. We also show that local infusion of InsP5 and InsP6 into a discrete brain stem nucleus implicated in cardiovascular regulation, results in dose-dependent changes in heart rate and blood pressure.     

Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum

Inositol 1,4,5-trisphosphate (InsP3) can initiate calcium release into the cytoplasm in a variety of cells. From experiments using permeabilized cells, membrane vesicles, and patch-clamp techniques, it has been suggested that InsP3 acts by directly opening calcium channels. Here, we show that InsP3 induced openings of channels in planar lipid bilayers into which vesicles made from aortic muscle sarcoplasmic reticulum (SR) were incorporated. Activation of channels by InsP3 was not observed when vesicles made from SR of cardiac or skeletal muscle were incorporated into planar lipid bilayers. The present study demonstrates for the first time unique properties of an InsP3-gated calcium channel in sarcoplasmic reticulum vesicles from vascular smooth muscle. This InsP3-activated channel from aortic SR differs strikingly from the calcium-gated calcium channel of striated muscle SR in single-channel conductance and pharmacology.     

珍珠鸟和家鸡甲状旁腺激素3型受体(PTH3R)的结构与表达图谱分析

甲状旁腺激素(PTH)与动物钙稳态调控和骨代谢平衡相关,其生理作用是通过甲状旁腺激素受体(PTHR)介导。甲状旁腺激素受体家族包括三个不同的亚型,其中甲状旁腺激素3型受体(PTH3R)在非哺乳类脊椎动物生长发育过程中起着重要作用,然而PTH3R在鸟类中的研究则相对较少。 本研究采用RT-PCR方法,首先克隆了珍珠鸟和家鸡的PTH3R基因全长cDNA序列。结果显示,家鸡PTH3R (cPTH3R) cDNA全长1632 bp,编码543个氨基酸,珍珠鸟PTH3R(zPTH3R-w) cDNA序列全长1563 bp,编码520个氨基酸,其蛋白均含有信号肽序列、七次跨膜区等特征性结构。此外,在珍珠鸟中还发现一个新剪接变体zPTH3R-v1,其cDNA序列全长1468 bp,编码488个氨基酸,其缺失了第3外显子进而导致第1跨膜结构域缺失。利用生物信息学方法,我们还对珍珠鸟和家鸡PTH3R蛋白序列进行三维建模。 采用RT-PCR方法,本研究也对珍珠鸟PTH3R基因进行组织表达分析。结果显示,zPTH3R及其剪切变体zPTH3R-v1在珍珠鸟脑及外周组织中广泛表达。珍珠鸟和家鸡PTH3R基因的成功克隆与结构解析,将为下一步开展PTH3R在鸟类中的功能研究奠定重要基础。