昆虫嗅觉相关蛋白的结构和功能

昆虫在长期进化的过程中形成了复杂的嗅觉系统,气味剂结合蛋白(odorant binding proteins,OBPs)、嗅觉受体(olfactory receptors,ORs)是其最主要的组分.其主要作用是结合外围挥发性的气味分子并将信号传递给细胞内的第二信使.OBPs和ORs的结构、功能、表达、进化是昆虫行为与进化关系的重要研究领域和研究热点.本文主要总结了近年来昆虫OBPs和ORs的结构特点、生理功能、表达特点、遗传进化等方面研究的最新进展,对OBPs和ORs的研究趋势进行了展望,为昆虫嗅觉系统进化研究及寻找害虫防治新途径提供参考信息.     

Structure and evolution of calcium-modulated proteins

This review suggests that the intracellular functions of calcium are best understood in terms of calcium's functioning as a second messenger. Further, when functioning as a second messenger, calcium completes its mission not by transferring charge nor by binding to lipid but by binding to specific targets, calcium-modulated proteins. This concept is broadly interpreted to include proteins involved in calcium transport. There is strong evidence that many, if not all, of these calcium-modulated proteins are homologs. Their structures and properties are contrasted to those of extracellular calcium-binding proteins which are not homologous to one another or to the intracellular calcium-modulated proteins. Finally, this line of thought leads to a suggestion of the evolutionary reason for the choice of calcium as the sole inorganic second messenger.     

The TRP family of channels: a complex gallery of characters

Calcium influxes are of fundamental importance in eukaryotic cell functions. These calcium influxes are carried by different classes of membrane proteins that allow regulated calcium entry. If in excitable cells, such as neurones or muscle, voltage-dependent calcium channels represent the main source of calcium influx, other proteins are needed to assume such a function in non-excitable cells. In these, a sustained calcium influx is observed, secondary to phospholipase C activation, IP3 synthesis and internal calcium release. The identity of proteins implicated in this second messenger calcium-driven influx, as well as the mechanisms of activation of these channels have long been debated. In recent years, genes encoding a new kind of cationic channels called TRP channels have been identified. This molecular work has set the basis for further functional studies and helped to gain crucial information on the mechanisms by which extracellular calcium can penetrate into non-excitable cells. This review will present the most recent advances obtained on the molecular diversity of TRP channels and their mode of gating.     

Differential regulation of proteins by bursting calcium oscillations--a theoretical study

Calcium in ionic form is a second messenger connecting several input signals to several target processes in the cell. The question arises how one second messenger can transmit more than one signal simultaneously (bow-tie structure of signalling). Experimental data on calcium dynamics often show patterns of successive low-peak and high-peak oscillatory phases, known as bursting. Here, we propose that bursting calcium oscillations can perform the function of simultaneous transmission of two signals at physiological calcium concentrations, for example, by selective activation of two calcium-binding proteins. This differential regulation by periodic bursting is investigated in a theoretical model. The two proteins are assumed to be activated by calcium, and one of them is assumed to be subject to biphasic regulation due to additional inhibitory binding sites. To explore which characteristics of the complex signal could be responsible for independent regulation of low-peak activated and spike activated targets, different bursting patterns of simplified square pulses are applied. Depending on the change in the bursting pattern, one protein can be gradually activated at a constant level of the other protein's activity, or the two proteins can be activated simultaneously, or one protein can be activated while the other one is deactivated simultaneously. Thus, the two proteins can be regulated virtually independently.     

Calmodulin‐mediated events during the life cycle of the amoebozoan Dictyostelium discoideum

This review focusses on the functions of intracellular and extracellular calmodulin, its target proteins and their binding proteins during the asexual life cycle of Dictyostelium discoideum. Calmodulin is a primary regulatory protein of calcium signal transduction that functions throughout all stages. During growth, it mediates autophagy, the cell cycle, folic acid chemotaxis, phagocytosis, and other functions. During mitosis, specific calmodulin‐binding proteins translocate to alternative locations. Translocation of at least one cell adhesion protein is calmodulin dependent. When starved, cells undergo calmodulin‐dependent chemotaxis to cyclic AMP generating a multicellular pseudoplasmodium. Calmodulin‐dependent signalling within the slug sets up a defined pattern and polarity that sets the stage for the final events of morphogenesis and cell differentiation. Transected slugs undergo calmodulin‐dependent transdifferentiation to re‐establish the disrupted pattern and polarity. Calmodulin function is critical for stalk cell differentiation but also functions in spore formation, events that begin in the pseudoplasmodium. The asexual life cycle restarts with the calmodulin‐dependent germination of spores. Specific calmodulin‐binding proteins as well as some of their binding partners have been linked to each of these events. The functions of extracellular calmodulin during growth and development are also discussed. This overview brings to the forefront the central role of calmodulin, working through its numerous binding proteins, as a primary downstream regulator of the critical calcium signalling pathways that have been well established in this model eukaryote. This is the first time the function of calmodulin and its target proteins have been documented through the complete life cycle of any eukaryote.     

Lymphokine signal transduction

Lymphokines are a group of signalling molecules involved in communication between cells, mainly those of the immune system. The lymphokines are multi-functional and most of them have mitogenic or co-mitogenic activity. An understanding of lymphokine biology is essential to understand how the immune system develops and functions and to provide a rationale for their use in immunotherapy.The potential to understand the cell biology of the lymphokines has recently become more apparent as molecular biological techniques have first of all produced recombinant factors and secondly have provided clues to the signal transduction pathways by cloning receptors, applying site-directed mutational analysis and also probing for specific promoters and enhancers that are activated along the signal pathway.This review discusses the information that has come from these recent analyses which blends with the biochemical analysis of the second messenger systems in an effort to understand the signalling pathways of the lymphokines.     

Local calcium signaling in neurons

Transient rises in the cytoplasmic concentration of calcium ions serve as second messenger signals that control many neuronal functions. Selective triggering of these functions is achieved through spatial localization of calcium signals. Several qualitatively different forms of local calcium signaling can be distinguished by the location of open calcium channels as well as by the distance between these channels and the calcium binding proteins that serve as the molecular targets of calcium action. Local calcium signaling is especially prominent at presynaptic active zones and postsynaptic densities, structures that are distinguished by highly organized macromolecular arrays that yield precise spatial arrangements of calcium signaling proteins. Similar forms of local calcium signaling may be employed throughout the nervous system, though much remains to be learned about the molecular underpinnings of these events.     

Cell signaling, beyond cytosolic calcium in eukaryotes

Calcium functions as a secondary messenger within the cytosols of eukaryotes. This serves as a reference point to evaluate three related questions:Exploration of these three interrelated questions indicates the importance of more sensitive techniques and of a refined concept of information transfer and transduction. 1. Calcium, as well as cyclic AMP, also functions as a paracrine messenger; how specific and extensive is this use? 2. Calcium binding proteins and calcium extrusion mechanisms have been identified in prokaryotes; does it function as a messenger? 3. The concentrations of other divalent cations, especially zinc and magnesium, are well regulated and perturbations have specific physiological impacts; are these divalents involved in information transfer? Exploration of these three interrelated questions indicates the importance of more sensitive techniques and of a refined concept of information transfer and transduction.     

Calcium receptor is functionally expressed in rat neonatal ventricular cardiomyocytes

Both intra- and extracellular calcium play multiple roles in the physiology and pathophysiology of cardiomyocytes, especially in stimulus-contraction coupling. The intracellular calcium level is closely controlled through the concerted actions of calcium channels, exchangers, and pumps; however, the expression and function(s) of the so-called calcium-sensing receptor (CaR) in the heart remain less well characterized. The CaR is a seven-transmembrane receptor, which, in response to noncovalent binding of extracellular calcium, activates intracellular effectors, including G proteins and extracellular signal-regulated kinases (ERK1/2). We have shown that cultured neonatal cardiomyocytes express the CaR messenger RNA and the CaR protein. Furthermore, increasing concentrations of extracellular calcium and a type II CaR activator "calcimimetic" caused inositol phosphate (IP) accumulation, downregulated tritiated thymidine incorporation, and supported ERK1/2 phosphorylation, suggesting that the CaR protein is functionally active. Interestingly, the calcimimetic induced a more rapid ERK1/2 phosphorylation than calcium and left-shifted the IP concentration-response curve for extracellular calcium, supporting the hypothesis that CaR is functionally expressed in cardiac myocytes. This notion was underscored by studies using a virus containing a dominant-negative CaR construct, because this protein blunted the calcium-induced IP response. In conclusion, we have shown that the CaR is functionally expressed in neonatal ventricular cardiomyocytes and that the receptor activates second messenger pathways, including IP and ERK, and decreases DNA synthesis. A specific calcium-sensing receptor on cardiac myocytes could play a role in regulating cardiac development, function, and homeostasis.     

Pharmacology of stomoxytachykinin receptor depends on second messenger system

STKR is a neurokinin receptor derived from the stable fly, Stomoxys calcitrans. Insect tachykinin-related peptides, also referred to as "insectatachykinins", produce dose-dependent calcium and cyclic AMP responses in cultured Drosophila melanogaster Schneider 2 (S2) cells that were stably transfected with the cloned STKR cDNA. Pronounced differences in pharmacology were observed between agonist-induced calcium and cyclic AMP responses. The results indicate that the pharmacological properties of STKR depend on its coupling to a unique second messenger system. Therefore, a model postulating the existence of multiple active receptor conformations is proposed. This article presents the first evidence that an insect peptide receptor with dual coupling properties to second messenger systems can display agonist-dependent functional differences.     

From ligand binding to gene expression: new insights into the regulation of G-protein-coupled receptors.

Transmembrane signaling systems relay information from the exterior to the interior of a cell, through a series of complex protein-protein interactions and second messenger cascades. One such system consists of the G-protein-coupled receptors, which interact with G proteins upon ligand binding, and in turn activate an effector molecule. The receptor is the first component in this signaling cascade and is subject to considerable regulation. Recent studies have shown that these regulatory events can occur at the levels of receptor protein modification and receptor gene expression. Interestingly, some of these processes appear to be mediated by the same second messenger systems that these receptors activate, which leads to various forms of positive and negative feedback regulation.     

Functional Conservation of Calreticulin in Euglena gracilis

Calreticulin is the major high capacity, low affinity Ca²⁺ binding protein localized within the endoplasmic reticulum. It functions as a reservoir for triggered release of Ca²⁺ by the endoplasmic reticulum and is thus integral to eukaryotic signal transduction pathways involving Ca²⁺ as a second messenger. The early branching photosynthetic protist Euglena gracilis is shown to possess calreticulin as its major high capacity Ca²⁺ binding protein. The protein was purified, microsequenced and cloned. Like its homologues from higher eukaryotes, calreticulin from Euglena possesses a short signal peptide for endoplasmic reticulum import and the C-terminal retention signal KDEL, indicating that these components of the eukaryotic protein routing apparatus were functional in their present form prior to divergence of the euglenozoan lineage. A gene phytogeny for calreticulin and calnexin sequences in the context of eukaryotic homologues indicates i) that these Ca²⁺ binding endoplasmic reticulum proteins descend from a gene duplication that occurred in the earliest stages of eukaryotic evolution and furthermore iii that Euglenozoa express the calreticulin protein of the kinetoplastid (trypanosomes and their relatives) lineage, rather than that of the eukaryotic chlorophyte which gave rise to Euglena's plastids. Evidence for conservation of endoplasmic reticulum routing and Ca²⁺ binding function of calreticulin from Euglena traces the functional history of Ca²⁺ second messenger signal transduction pathways deep into eukaryotic evolution.     

Moonlighting is mainstream: paradigm adjustment required

Moonlighting--the performance of more than one function by a single protein--is becoming recognized as a common phenomenon with important implications for systems biology and human health. The different functions of a moonlighting protein may use different regions of the protein structure, or alternative structures that occur due to post-translational modifications and/or differences in binding partners. Often the different functions of moonlighting proteins are used at different times or in different places. The existence of moonlighting functions complicates efforts to understand metabolic and regulatory networks, as well as physiological and pathological processes in organisms. Because moonlighting functions can play important roles in disease processes, an improved understanding of moonlighting proteins will provide new opportunities for pharmacological manipulations that specifically target a function involved in pathology while sparing physiologically important functions.     

The role of loops 3 and 4 in the interdomains and intersubunits communication of E. coli cAMP receptor protein

Cyclic AMP serves as an intracellular messenger in cells and regulates a variety of biological functions by transmitting information through proteins. These proteins of different functions all consist of a cAMP-binding motif, and the structure of this motif is highly conserved with an exception of the loop 3 and 4. In current study, cAMP receptor protein was employed as a model system to investigate the function of the two loops. The results indicated that the loop 3 involves in the intersubunits communication of CRP, whereas the loop 4 involves in cAMP binding and interdomains communication.     

The Rat Neurotensin Receptor Expressed in Chinese Hamster Ovary Cells Mediates the Release of Inositol Phosphates

To study second messenger synthesis mediated by the cloned rat neurotensin receptor, we derived a cell line stably expressing this receptor. The cDNA clone of this receptor was subcloned into the pcDNA1neo expression vector. This construct was then used to transfect Chinese hamster ovary (CHO)-K1 cells. Colony clones, selected for resistance to antibiotic G-418 sulfate, were isolated and grown separately. Nineteen individual clones were screened for total [3H]neurotensin binding as an indication of neurotensin receptor expression. The clone (CHO-rNTR-10) showing the highest level of specific [3H]neurotensin binding was characterized further. With intact cells, the equilibrium dissociation constant (KD) for specific [3H]neurotensin binding was 18 nM, and the maximal number of binding sites (Bmax) was 900 fmol/mg of protein or 740 fmol/10(6) cells (approximately 4.4 x 10(5) sites on the cellular surface). Whereas the KD was similar to that found in other cellular systems, for example, the murine neuroblastoma clone N1E-115, the Bmax exceeded previously reported values. Incubation of intact CHO-rNTR-10 cells with neurotensin caused the release of inositol phosphates in a dose-dependent manner (EC50 = 3 nM), results indicating that the expressed transfected receptor was functional. Neurotensin did not inhibit cyclic AMP levels stimulated by forskolin. As with other systems, neurotensin (8-13) was more potent than neurotensin Neurotensin-mediated inositol phosphate release is the first report of second messenger synthesis for this receptor expressed in a transfected cell line. These results suggest that the relation between structure and function of the neurotensin receptor can be readily studied in transfected cell lines.     

Advances of calcium signals involved in plant anti-drought

Considerable progresses have taken place, both in the methodology available to study changes in intracellular cytosolic calcium and in our understanding of calcium signaling cascades, but how calcium signals function in plant drought resistance is questionable. In plant cells, calcium plays roles as a second messenger coupling a wide range of extracellular stimuli with intracellular responses. Different extracellular stimuli trigger specific calcium signatures: dynamics, amplitude and duration of calcium transients specify the nature, implication and intensity of stimuli. Calcium-binding proteins (sensors) play a critical role in decoding calcium signatures and transducing signals by activating specific targets and corresponding metabolic pathways. Calmodulin is a calcium sensor known to regulate the activity of many mammalian proteins, whose targets in plants are now being identified. Higher plants possess a rapidly growing list of calmodulin targets with a variety of cellular functions. Nevertheless, many targets appear to be unique to higher plants and remain characterized, calling for a concerted effort to elucidate their functions. To date, three major classes of plant calcium signals, including calcium permeable ion channels, Ca(2+)/H(+) antiporters and Ca(2+)-ATPases, have been responsible for drought-stress signal transduction. This review summarizes the current knowledge of calcium signals involved in plant anti-drought and plant water use efficiency (WUE) and presents suggestions for future focus of study.     

钙离子结合蛋白及其在神经系统疾病中的作用

钙离子作为第二信使参与多种途径的调控,钙离子结合蛋白在此过程中起着重要的作用．通过对钙离子结合蛋白的分布特征、结构分析,新的成员以及新的功能不断地被发现．在疾病发生过程中,钙离子结合蛋白动态平衡的破坏与线粒体的异常、自由基的损害有密切的关系,并已在多种疾病特别是神经系统的疾病中得到了证实．本文就钙离子结合蛋白的特征以及在主要神经系统疾病中作用的研究进展进行简要综述．     

The ordered visual transduction complex of the squid photoreceptor membrane

The study of visual transduction has given invaluable insight into the mechanisms of signal transduction by heptahelical receptors that act via guanine nucleotide binding proteins (G-proteins). However, the cyclic-GMP second messenger system seen in vertebrate photoreceptor cells is not widely used in other cell types. In contrast, the retina of higher invertebrates, such as squid, offers an equally accessible transduction system, which uses the widespread second messenger chemistry of an increase in cytosolic calcium caused by the production of inositol-(1,4,5)-trisphosphate (InsP3) by the enzyme phospholipase C, and which may be a model for store-operated calcium influx. In this article, we highlight some key aspects of invertebrate visual transduction as elucidated from the combination of biochemical techniques applied to cephalopods, genetic techniques applied to flies, and electrophysiology applied to the horseshoe crab. We discuss the importance and applicability of ideas drawn from these model systems to the understanding of some general processes in signal transduction, such as the integration of the cytoskeleton into the signal transduction process and the possible modes of regulation of store-operated calcium influx.     

Solution structure of a novel calcium binding protein, MTH1880, from Methanobacterium thermoautotrophicum

MTH1880 is a hypothetical protein from Methanobacterium thermoautotrophicum, a target organism of structural genomics. The solution structure determined by NMR spectroscopy demonstrates a typical alpha + beta-fold found in many proteins with different functions. The molecular surface of the protein reveals a small, highly acidic pocket comprising loop B (Asp36, Asp37, Asp38), the end of beta2 (Glu39), and loop D (Ser57, Ser58, Ser61), indicating that the protein would have a possible cation binding site. The NMR resonances of several amino acids within the acidic binding pocket in MTH1880, shifted upon addition of calcium ion. This calcium binding motif and overall topology of MTH1880 differ from those of other calcium binding proteins. MTH1880 did not show a calcium-induced conformational change typical of calcium sensor proteins. Therefore, we propose that the MTH1880 protein contains a novel motif for calcium-specific binding, and may function as a calcium buffering protein.