The crystal structure of the complex of PII and acetylglutamate kinase reveals how PII controls the storage of nitrogen as arginine

Photosynthetic organisms can store nitrogen by synthesizing arginine, and, therefore, feedback inhibition of arginine synthesis must be relieved in these organisms when nitrogen is abundant. This relief is accomplished by the binding of the PII signal transduction protein to acetylglutamate kinase (NAGK), the controlling enzyme of arginine synthesis. Here, we describe the crystal structure of the complex between NAGK and PII of Synechococcus elongatus, at 2.75-A resolution. We prove the physiological relevance of the observed interactions by site-directed mutagenesis and functional studies. The complex consists of two polar PII trimers sandwiching one ring-like hexameric NAGK (a trimer of dimers) with the threefold axes of these molecules aligned. The binding of PII favors a narrow ring conformation of the NAGK hexamer that is associated with arginine sites having low affinity for this inhibitor. Each PII subunit contacts one NAGK subunit only. The contacts map in the inner circumference of the NAGK ring and involve two surfaces of the PII subunit. One surface is on the PII body and interacts with the C-domain of the NAGK subunit, helping widen the arginine site found on the other side of this domain. The other surface is at the distal region of a protruding large loop (T-loop) that presents a novel compact shape. This loop is inserted in the interdomain crevice of the NAGK subunit, contacting mainly the N-domain, and playing key roles in anchoring PII on NAGK, in activating NAGK, and in complex formation regulation by MgATP, ADP, 2-oxoglutarate, and by phosphorylation of serine-49.     

蛋白酪氨酸磷酸酶1B与细胞信号转导

蛋白酪氨酸磷酸酶-1B(PTP-1B)通过对细胞内不同蛋白底物脱磷酸化参与不同的生理反应,在胰岛素、瘦素等多条细胞信号通路中发挥作用.因其参与多种生理、病理过程,近年来PTP-1B抑制剂的提取和研制成为相关疾病防治的新方向.     

Tau蛋白调控Fyn信号通路在阿尔茨海默病大鼠发病机制中的作用

目的 探讨Tau蛋白调控Fyn信号通路在阿尔茨海默病(AD)大鼠发病中的作用机制.方法 雄性SD大鼠60只随机分为正常组,模型组及观察组.采用β淀粉样蛋白毒性片段(Aβ25～35)海马注射制备AD大鼠模型.10 μmol/L SB216763(Tau蛋白抑制剂)注射至观察组大鼠脑组织.采用SABC法对大鼠脑组织中Tau蛋白进行免疫组化检测,Western印迹检测Fyn和Fak的表达.结果 与正常组相比,模型组大鼠脑组织Tau蛋白含量及Fyn和Fak的表达显著增加(P＜0.05);与模型组相比,观察组Tau蛋白含量及Fyn和Fak的表达显著增加(P＜0.05).结论 Tau蛋白可能通过调控Fyn信号通路参与了AD的发病过程,为AD的治疗提供了新的研究方向.     

Hedgehog signal transduction by Smoothened: Pharmacologic evidence for a 2-step activation process

The Hedgehog (Hh) signaling pathway controls growth, cell fate decisions, and morphogenesis during development. Damage to Hh transduction machinery can lead to birth defects and cancer. The transmembrane protein Smoothened (Smo) relays the Hh signal and is an important drug target in cancer. Smo enrichment in primary cilia is thought to drive activation of target genes. Using small-molecule agonists and antagonists to dissect Smo function, we find that Smo enrichment in cilia is not sufficient for signaling and a distinct second step is required for full activation. This 2-step mechanism—localization followed by activation—has direct implications for the design and use of anticancer therapeutics targeted against Smo.     

Integration of calcium with the signaling network in cardiac myocytes

Calcium has evolved as global intracellular messenger for signal transduction in the millisecond time range by reversibly binding to calcium-sensing proteins. In the cardiomyocyte, ion pumps, ion exchangers and channels keep the cytoplasmic calcium level at rest around approximately 100 nM which is more than 10,000-fold lower than outside the cell. Intracellularly, calcium is mainly stored in the sarcoplasmic reticulum, which comprises the bulk of calcium available for the heartbeat. Regulation of cardiac function including contractility and energy production relies on a three-tiered control system, (i) immediate and fast feedback in response to mechanical load on a beat-to-beat basis (Frank-Starling relation), (ii) more sustained regulation involving transmitters and hormones as primary messengers, and (iii) long-term adaptation by changes in the gene expression profile. Calcium signaling over largely different time scales requires its integration with the protein kinase signaling network which is governed by G-protein-coupled receptors, growth factor and cytokine receptors at the surface membrane. Short-term regulation is dominated by the beta-adrenergic system, while long-term regulation with phenotypic remodeling depends on sustained signaling by growth factors, cytokines and calcium. Mechanisms and new developments in intracellular calcium handling and its interrelation with the MAPK signaling pathways are discussed in detail.     

上海市崇明区老年人成纤维活化蛋白与代谢综合征的相关性分析

目的 探讨老年人血浆成纤维活化蛋白(FAP)水平与代谢综合征的相关性.方法 2011年6月-12月,采取随机整群抽样的方法,以上海市崇明区城桥镇587例常住居民为研究对象,其中男性202例、女性385例.通过问卷调查和体格检查采集人体基本参数,采集标本完成相关实验室检查,分析FAP水平与代谢综合征的相关性.结果 男性血...     

Iron conservation by reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii

The marine nitrogen fixing microorganisms (diazotrophs) are a major source of nitrogen to open ocean ecosystems and are predicted to be limited by iron in most marine environments. Here we use global and targeted proteomic analyses on a key unicellular marine diazotroph Crocosphaera watsonii to reveal large scale diel changes in its proteome, including substantial variations in concentrations of iron metalloproteins involved in nitrogen fixation and photosynthesis, as well as nocturnal flavodoxin production. The daily synthesis and degradation of enzymes in coordination with their utilization results in a lowered cellular metalloenzyme inventory that requires ～40% less iron than if these enzymes were maintained throughout the diel cycle. This strategy is energetically expensive, but appears to serve as an important adaptation for confronting the iron scarcity of the open oceans. A global numerical model of ocean circulation, biogeochemistry and ecosystems suggests that Crocosphaera's ability to reduce its iron-metalloenzyme inventory provides two advantages: It allows Crocosphaera to inhabit regions lower in iron and allows the same iron supply to support higher Crocosphaera biomass and nitrogen fixation than if they did not have this reduced iron requirement.     

CLE-CLAVATA1 peptide-receptor signaling module regulates the expansion of plant root systems in a nitrogen-dependent manner

Morphological plasticity of root systems is critically important for plant survival because it allows plants to optimize their capacity to take up water and nutrients from the soil environment. Here we show that a signaling module composed of nitrogen (N)-responsive CLE (CLAVATA3/ESR-related) peptides and the CLAVATA1 (CLV1) leucine-rich repeat receptor-like kinase is expressed in the root vasculature in Arabidopsis thaliana and plays a crucial role in regulating the expansion of the root system under N-deficient conditions. CLE1, -3, -4, and -7 were induced by N deficiency in roots, predominantly expressed in root pericycle cells, and their overexpression repressed the growth of lateral root primordia and their emergence from the primary root. In contrast, clv1 mutants showed progressive outgrowth of lateral root primordia into lateral roots under N-deficient conditions. The clv1 phenotype was reverted by introducing a CLV1 promoter-driven CLV1:GFP construct producing CLV1:GFP fusion proteins in phloem companion cells of roots. The overaccumulation of CLE2, -3, -4, and -7 in clv1 mutants suggested the amplitude of the CLE peptide signals being feedback-regulated by CLV1. When CLE3 was overexpressed under its own promoter in wild-type plants, the length of lateral roots was negatively correlated with increasing CLE3 mRNA levels; however, this inhibitory action of CLE3 was abrogated in the clv1 mutant background. Our findings identify the N-responsive CLE-CLV1 signaling module as an essential mechanism restrictively controlling the expansion of the lateral root system in N-deficient environments.Living organisms have developed dynamic strategies to explore nutrients in the environment. Morphological plasticity of plant roots and microorganisms is often compared with foraging behavior of animals. Plant roots are highly dynamic systems because they can modify their structure to reach nutrient resources in soil and optimize their nutrient uptake capacities. This strategy appears to be associated with morphological adaptation, because plants are sessile in nature and nutrient availabilities in soil are often altered by surrounding biotic and abiotic factors and climate changes. Morphological modifications of plant root systems are particularly prominent when they grow in soil environments with unbalanced nutrient availabilities (1–4). Among the essential elements required for plant growth, nitrogen (N) has a particularly strong effect on root development (1–6). Lateral roots can be developed in N-rich soil patches where adequate amounts of nitrate (NO₃⁻) or ammonium (NH₄⁺) are available, whereas this local outgrowth of lateral roots is restricted in N-deficient patches (7–9). In addition to these local N responses, lateral root growth is stimulated in response to mild N deficiency and suppressed under excess N supply by systemic plant signals carrying information on the nutritional status of distant plant organs (4, 10–13). These morphological responses are important for plant fitness and N acquisition, despite the cost for structuring the root system architecture (2, 6). However, lateral root growth is not sustained when plants are deprived of N for an extended period (4). Under such severe circumstances, the development of new lateral roots should rather be restricted to prevent the risk of extending roots into N-poor environments. Economizing the cost for root development appears to be an important morphological strategy for plant survival.To modify root traits in response to changing N availabilities, plants use various types of signaling molecules including hormones and small RNAs (10, 13–17). In particular, auxin signaling proteins and auxin transporters have been proven essential for lateral root development in response to local nitrate supplies (10, 14–17). These proteins are involved in increasing auxin sensitivity or auxin accumulation at lateral root initials or lateral root tips exposed to NO₃⁻, and the NRT1.1 nitrate transporter has been suggested to play a key role in NO₃⁻ sensing (8, 17, 18). In addition, mutations of the nitrate transporter NRT2.1 have been shown to repress or stimulate lateral root initiation depending on N conditions and sucrose supply (12, 19). Thus, N-dependent root development is apparently under control of complex mechanisms, although its signaling components have remained largely unidentified. In this study, we have identified several homologs of the CLE (CLAVATA3/ESR-related) gene family (20–24) to be up-regulated by N deficiency and involved in this yet unresolved regulatory mechanism. CLAVATA3 (CLV3) is known as a signaling peptide that binds to the CLAVATA1 (CLV1) leucine-rich repeat receptor-like kinase (LRR-RLK) and controls stem cell differentiation in the shoot apical meristem (25–32). CLE-receptor signaling modules are also known to control meristem function in the primary and lateral roots (33–35). The N-responsive CLE peptides described in the present study belong to the group of CLE peptides with the highest sequence similarity to CLAVATA3 (CLV3) (21–23) and may partly substitute for CLV3 in the shoot apical meristem (31, 36, 37). Our present findings indicate that the N-responsive CLE peptides and CLV1 are signaling components required for translating an N-deficient nutritional status into a morphological response inhibiting the outgrowth of lateral root primordia in Arabidopsis. The present study demonstrates a unique function of the CLE-CLV1 signaling module in roots and provides new insights into signaling mechanisms regulating the expansion of the plant root system in N-deficient environments.     

Tsukushi functions as a Wnt signaling inhibitor by competing with Wnt2b for binding to transmembrane protein Frizzled4

The Wnt signaling pathway is essential for the development of diverse tissues during embryogenesis. Signal transduction is activated by the binding of Wnt proteins to the type I receptor low-density lipoprotein receptor-related protein 5/6 and the seven-pass transmembrane protein Frizzled (Fzd), which contains a Wnt-binding site in the form of a cysteine-rich domain. Known extracellular antagonists of the Wnt signaling pathway can be subdivided into two broad classes depending on whether they bind primarily to Wnt or to low-density lipoprotein receptor-related protein 5/6. We show that the secreted protein Tsukushi (TSK) functions as a Wnt signaling inhibitor by binding directly to the cysteine-rich domain of Fzd4 with an affinity of 2.3 × 10(-10) M and competing with Wnt2b. In the developing chick eye, TSK is expressed in the ciliary/iris epithelium, whereas Wnt2b is expressed in the adjacent anterior rim of the optic vesicle, where it controls the differentiation of peripheral eye structures, such as the ciliary body and iris. TSK overexpression effectively antagonizes Wnt2b signaling in chicken embryonic retinal cells both in vivo and in vitro and represses Wnt-dependent specification of peripheral eye fates. Conversely, targeted inactivation of the TSK gene in mice causes expansion of the ciliary body and up-regulation of Wnt2b and Fzd4 expression in the developing peripheral eye. Thus, we uncover a crucial role for TSK as a Wnt signaling inhibitor that regulates peripheral eye formation.     

T-lymphocyte interleukin 2-dependent tyrosine protein kinase signal transduction involves the activation of p56lck.

Addition of interleukin 2 (IL-2) to IL-2-dependent T cells results in tyrosine protein kinase signal transduction events even though the IL-2 receptor alpha and beta chains lack intrinsic enzymatic activity. Here we report that addition of IL-2 to IL-2-dependent human T cells transiently stimulates the specific activity of p56lck, a member of the src family of nonreceptor tyrosine protein kinases expressed at high levels in T lymphocytes. The ability of IL-2 to induce p56lck activation was found to be independent of the capacity of p56lck to associate with either CD4 or CD8. Following IL-2 treatment, p56lck was found to undergo serine/threonine phosphorylation modifications that resulted in altered mobility of the lck gene product on polyacrylamide gels. These observations raise the possibility that p56lck participates in IL-2-mediated signal transduction events in T cells.     

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+

The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg²⁺. Here we investigated the active forms of ATP and found that the action of MgATP²⁻ and ATP⁴⁻ differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP²⁻ promotes opening with very low efficacy. In contrast, both free ATP and MgATP²⁻ robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg²⁺ to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP²⁻ and weak regulation by Mg²⁺. These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP²⁻ and regulation by Mg²⁺, and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons.Seven subtypes of P2X receptors have been identified in mammals that can form either homomeric (P2X1, P2X2, P2X3, P2X4, P2X5, P2X7) or heteromeric (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/5, P2X2/6, P2X4/6, and possibly, P2X4/7) channels (1–8). These subtypes of P2X receptors have distinct gating properties, pharmacology, and cellular distributions. P2X1 and P2X3 receptors desensitize within a few hundred milliseconds when opened by ATP, and their distributions are restricted to either smooth muscle cells and platelets (P2X1) or a subset of sensory neurons (P2X3) (1, 9–14). P2X2 and P2X4 receptors exhibit slow desensitization during prolonged ATP application, and these receptors are the most abundant subtypes in the central nervous system (15). P2X2 subunits also express in a subset of sensory neurons; however, in these cells they only form heteromeric channels with P2X3 subunits (12, 16, 17). In sensory neurons, P2X3 homomeric channels together with P2X2/3 heteromeric channels play important roles in mediating the primary sensory effects of ATP, and knock-out animals with either P2X3 deletion or P2X2 and P2X3 double-deletions have revealed critical roles in taste, pain, oxygen sensing, and bladder filling (17–20).A long-standing conundrum in P2X receptor-mediated signaling concerns the forms of ATP that activate these channels. In neutral solutions, ATP is ionized and exists mostly as free ATP (ATP⁴⁻), an efficient chelator of divalent cations such as Mg²⁺, and to a lesser extent Ca²⁺ (21). In extracellular biological compartments, such as the synaptic cleft, Ca²⁺ and Mg²⁺ are present in the millimolar range, and therefore only a relatively small fraction of ATP released from vesicles is present in the free form. Although a range of important studies have explored the regulatory effects of Ca²⁺ and Mg²⁺ on P2X receptor channels (22–30), the essential question of which forms of ATP serve as agonists remains unresolved. Several previous studies have reported that P2X2, P2X7, and the native P2X receptors in cilia are activated by ATP in solutions containing low concentrations of divalent cations, and that the addition of divalent cations shifts the concentration dependence for activation of the channels to higher ATP concentrations, suggesting that either ATP⁴⁻ is the most active form of ATP or that divalent cations regulate those subtypes through allosteric mechanisms (27, 30–36). In the present study, we investigated the form(s) of ATP that serve as agonists for a range of subtypes of P2X receptor channels. Our primary focus was to determine whether ATP⁴⁻ or MgATP²⁻ are the principal agonists and to explore whether Mg²⁺ might serve specific regulatory roles. Our results demonstrate that the action of MgATP²⁻ and ATP⁴⁻ differ between subtypes of P2X receptors, and reveal that heteromeric channels can have unique hybrid phenotypes, findings that will be crucial for understanding the physiological functions of these channels in both the peripheral and central nervous systems.