Single Nucleotide Polymorphisms of Human STING Can Affect Innate Immune Response to Cyclic Dinucleotides

The STING (stimulator of interferon genes) protein can bind cyclic dinucleotides to activate the production of type I interferons and inflammatory cytokines. The cyclic dinucleotides can be bacterial second messengers c-di-GMP and c-di-AMP, 3’5’-3’5’ cyclic GMP-AMP (3’3’ cGAMP) produced by Vibrio cholerae and metazoan second messenger 2’5’-3’5’ Cyclic GMP-AMP (2’3’ cGAMP). Analysis of single nucleotide polymorphism (SNP) data from the 1000 Genome Project revealed that R71H-G230A-R293Q (HAQ) occurs in 20.4%, R232H in 13.7%, G230A-R293Q (AQ) in 5.2%, and R293Q in 1.5% of human population. In the absence of exogenous ligands, the R232H, R293Q and AQ SNPs had only modest effect on the stimulation of IFN-β and NF-κB promoter activities in HEK293T cells, while HAQ had significantly lower intrinsic activity. The decrease was primarily due to the R71H substitution. The SNPs also affected the response to the cyclic dinucleotides. In the presence of c-di-GMP, the R232H variant partially decreased the ability to activate IFN-βsignaling, while it was defective for the response to c-di-AMP and 3’3’ cGAMP. The R293Q dramatically decreased the stimulatory response to all bacterial ligands. Surprisingly, the AQ and HAQ variants maintained partial abilities to activate the IFN-β signaling in the presence of ligands due primarily to the G230A substitution. Biochemical analysis revealed that the recombinant G230A protein could affect the conformation of the C-terminal domain of STING and the binding to c-di-GMP. Comparison of G230A structure with that of WT revealed that the conformation of the lid region that clamps onto the c-di-GMP was significantly altered. These results suggest that hSTING variation can affect innate immune signaling and that the common HAQ haplotype expresses a STING protein with reduced intrinsic signaling activity but retained the ability to response to bacterial cyclic dinucleotides.     

植物水分胁迫信号识别与转导

植物对水分胁迫信号作出反应,需要经过信号的识别,转导和胞间信使传递等过程,该文从胁迫感觉,第二信使系统及蛋白质可逆磷酸化等方面,介绍了植物细胞对水分胁迫（原初 ）信号和胁迫信号分子（脱落酸）识别转导的研究进展。     

Cyclic Nucleotide Gated Channels 7 and 8 Are Essential for Male Reproductive Fertility

The Arabidopsis thaliana genome contains 20 CNGCs, which are proposed to encode cyclic nucleotide gated, non-selective, Ca²⁺-permeable ion channels. CNGC7 and CNGC8 are the two most similar with 74% protein sequence identity, and both genes are preferentially expressed in pollen. Two independent loss-of-function T-DNA insertions were identified for both genes and used to generate plant lines in which only one of the two alleles was segregating (e.g., cngc7-1+/−/cngc8-2−/− and cngc7-3−/−/cngc8-1+/−). While normal pollen transmission was observed for single gene mutations, pollen harboring mutations in both cngc7 and 8 were found to be male sterile (transmission efficiency reduced by more than 3000-fold). Pollen grains harboring T-DNA disruptions of both cngc7 and 8 displayed a high frequency of bursting when germinated in vitro. The male sterile defect could be rescued through pollen expression of a CNGC7 or 8 transgene including a CNGC7 with an N-terminal GFP-tag. However, rescue efficiencies were reduced ∼10-fold when the CNGC7 or 8 included an F to W substitution (F589W and F624W, respectively) at the junction between the putative cyclic nucleotide binding-site and the calmodulin binding-site, identifying this junction as important for proper functioning of a plant CNGC. Using confocal microscopy, GFP-CNGC7 was found to preferentially localize to the plasma membrane at the flanks of the growing tip. Together these results indicate that CNGC7 and 8 are at least partially redundant and provide an essential function at the initiation of pollen tube tip growth.     

Bacterial Signal Transduction by Cyclic Di-GMP and Other Nucleotide Second Messengers

The first International Symposium on c-Di-GMP Signaling in Bacteria (22 to 25 March 2015, Harnack-Haus, Berlin, Germany) brought together 131 molecular microbiologists from 17 countries to discuss recent progress in our knowledge of bacterial nucleotide second messenger signaling. While the focus was on signal input, synthesis, degradation, and the striking diversity of the modes of action of the current second messenger paradigm, i.e., cyclic di-GMP (c-di-GMP), “classics” like cAMP and (p)ppGpp were also presented, in novel facets, and more recent “newcomers,” such as c-di-AMP and c-AMP-GMP, made an impressive appearance. A number of clear trends emerged during the 30 talks, on the 71 posters, and in the lively discussions, including (i) c-di-GMP control of the activities of various ATPases and phosphorylation cascades, (ii) extensive cross talk between c-di-GMP and other nucleotide second messenger signaling pathways, and (iii) a stunning number of novel effectors for nucleotide second messengers that surprisingly include some long-known master regulators of developmental pathways. Overall, the conference made it amply clear that second messenger signaling is currently one of the most dynamic fields within molecular microbiology, with major impacts in research fields ranging from human health to microbial ecology.     

Leaf Senescence Signaling: The Ca2+-Conducting Arabidopsis Cyclic Nucleotide Gated Channel2 Acts through Nitric Oxide to Repress Senescence Programming

Ca²⁺ and nitric oxide (NO) are essential components involved in plant senescence signaling cascades. In other signaling pathways, NO generation can be dependent on cytosolic Ca²⁺. The Arabidopsis (Arabidopsis thaliana) mutant dnd1 lacks a plasma membrane-localized cation channel (CNGC2). We recently demonstrated that this channel affects plant response to pathogens through a signaling cascade involving Ca²⁺ modulation of NO generation; the pathogen response phenotype of dnd1 can be complemented by application of a NO donor. At present, the interrelationship between Ca²⁺ and NO generation in plant cells during leaf senescence remains unclear. Here, we use dnd1 plants to present genetic evidence consistent with the hypothesis that Ca²⁺ uptake and NO production play pivotal roles in plant leaf senescence. Leaf Ca²⁺ accumulation is reduced in dnd1 leaves compared to the wild type. Early senescence-associated phenotypes (such as loss of chlorophyll, expression level of senescence-associated genes, H₂O₂ generation, lipid peroxidation, tissue necrosis, and increased salicylic acid levels) were more prominent in dnd1 leaves compared to the wild type. Application of a Ca²⁺ channel blocker hastened senescence of detached wild-type leaves maintained in the dark, increasing the rate of chlorophyll loss, expression of a senescence-associated gene, and lipid peroxidation. Pharmacological manipulation of Ca²⁺ signaling provides evidence consistent with genetic studies of the relationship between Ca²⁺ signaling and senescence with the dnd1 mutant. Basal levels of NO in dnd1 leaf tissue were lower than that in leaves of wild-type plants. Application of a NO donor effectively rescues many dnd1 senescence-related phenotypes. Our work demonstrates that the CNGC2 channel is involved in Ca²⁺ uptake during plant development beyond its role in pathogen defense response signaling. Work presented here suggests that this function of CNGC2 may impact downstream basal NO production in addition to its role (also linked to NO signaling) in pathogen defense responses and that this NO generation acts as a negative regulator during plant leaf senescence signaling.Senescence can be considered as the final stage of a plant’s development. During this process, nutrients will be reallocated from older to younger parts of the plant, such as developing leaves and seeds. Leaf senescence has been characterized as a type of programmed cell death (PCD; Gan and Amasino, 1997; Quirino et al., 2000; Lim et al., 2003). During senescence, organelles such as chloroplasts will break down first. Biochemical changes will also occur in the peroxisome during this process. When the chloroplast disassembles, it is easily observed as a loss of chlorophyll. Mitochondria, the source of energy for cells, will be the last cell organelles to undergo changes during the senescence process (Quirino et al., 2000). At the same time, other catabolic events (e.g. protein and lipid breakdown, etc.) are occurring (Quirino et al., 2000). Hormones may also contribute to this process (Gepstein, 2004). From this information we can infer that leaf senescence is regulated by many signals.Darkness treatment can induce senescence in detached leaves (Poovaiah and Leopold, 1973; Chou and Kao, 1992; Weaver and Amasino, 2001; Chrost et al., 2004; Guo and Crawford, 2005; Ülker et al., 2007). Ca²⁺ can delay the senescence of detached leaves (Poovaiah and Leopold, 1973) and leaf senescence induced by methyl jasmonate (Chou and Kao, 1992); the molecular events that mediate this effect of Ca²⁺ are not well characterized at present.Nitric oxide (NO) is a critical signaling molecule involved in many plant physiological processes. Recently, published evidence supports NO acting as a negative regulator during leaf senescence (Guo and Crawford, 2005; Mishina et al., 2007). Abolishing NO generation in either loss-of-function mutants (Guo and Crawford, 2005) or transgenic Arabidopsis (Arabidopsis thaliana) plants expressing NO degrading dioxygenase (NOD; Mishina et al., 2007) leads to an early senescence phenotype in these plants compared to the wild type. Corpas et al. (2004) showed that endogenous NO is mainly accumulated in vascular tissues of pea (Pisum sativum) leaves. This accumulation is significantly reduced in senescing leaves (Corpas et al., 2004). Corpas et al. (2004) also provided evidence that NO synthase (NOS)-like activity (i.e. generation of NO from l-Arg) is greatly reduced in senescing leaves. Plant NOS activity is regulated by Ca²⁺/calmodulin (CaM; Delledonne et al., 1998; Corpas et al., 2004, 2009; del Río et al., 2004; Valderrama et al., 2007; Ma et al., 2008). These studies suggest a link between Ca²⁺ and NO that could be operating during senescence.In animal cells, all three NOS isoforms require Ca²⁺/CaM as a cofactor (Nathan and Xie, 1994; Stuehr, 1999; Alderton et al., 2001). Notably, animal NOS contains a CaM binding domain (Stuehr, 1999). It is unclear whether Ca²⁺/CaM can directly modulate plant NOS or if Ca²⁺/CaM impacts plant leaf development/senescence through (either direct or indirect) effects on NO generation. However, recent studies from our lab suggest that Ca²⁺/CaM acts as an activator of NOS activity in plant innate immune response signaling (Ali et al., 2007; Ma et al., 2008).Although Arabidopsis NO ASSOCIATED PROTEIN1 (AtNOA1; formerly named AtNOS1) was thought to encode a NOS enzyme, no NOS-encoding gene has yet been identified in plants (Guo et al., 2003; Crawford et al., 2006; Zemojtel et al., 2006). However, the AtNOA1 loss-of-function mutant does display reduced levels of NO generation, and several groups have used the NO donor sodium nitroprusside (SNP) to reverse some low-NO related phenotypes in Atnoa1 plants (Guo et al., 2003; Bright et al., 2006; Zhao et al., 2007). Importantly, plant endogenous NO deficiency (Guo and Crawford, 2005; Mishina et al., 2007) or abscisic acid/methyl jasmonate (Hung and Kao, 2003, 2004) induced early senescence can be successfully rescued by application of exogenous NO. Addition of NO donor can delay GA-elicited PCD in barley (Hordeum vulgare) aleurone layers as well (Beligni et al., 2002).It has been suggested that salicylic acid (SA), a critical pathogen defense metabolite, can be increased in natural (Morris et al., 2000; Mishina et al., 2007) and transgenic NOD-induced senescent Arabidopsis leaves (Mishina et al., 2007). Pathogenesis related gene1 (PR1) expression is up-regulated in transgenic Arabidopsis expressing NOD (Mishina et al., 2007) and in leaves of an early senescence mutant (Ülker et al., 2007).Plant cyclic nucleotide gated channels (CNGCs) have been proposed as candidates to conduct extracellular Ca²⁺ into the cytosol (Sunkar et al., 2000; Talke et al., 2003; Lemtiri-Chlieh and Berkowitz, 2004; Ali et al., 2007; Demidchik and Maathuis, 2007; Frietsch et al., 2007; Kaplan et al., 2007; Ma and Berkowitz, 2007; Urquhart et al., 2007; Ma et al., 2009a, 2009b). Arabidopsis “defense, no death” (dnd1) mutant plants have a null mutation in the gene encoding the plasma membrane-localized Ca²⁺-conducting CNGC2 channel. This mutant also displays no hypersensitive response to infection by some pathogens (Clough et al., 2000; Ali et al., 2007). In addition to involvement in pathogen-mediated Ca²⁺ signaling, CNGC2 has been suggested to participate in the process of leaf development/senescence (Köhler et al., 2001). dnd1 mutant plants have high levels of SA and expression of PR1 (Yu et al., 1998), and spontaneous necrotic lesions appear conditionally in dnd1 leaves (Clough et al., 2000; Jirage et al., 2001). Endogenous H₂O₂ levels in dnd1 mutants are increased from wild-type levels (Mateo et al., 2006). Reactive oxygen species molecules, such as H₂O₂, are critical to the PCD/senescence processes of plants (Navabpour et al., 2003; Overmyer et al., 2003; Hung and Kao, 2004; Guo and Crawford, 2005; Zimmermann et al., 2006). Here, we use the dnd1 mutant to evaluate the relationship between leaf Ca²⁺ uptake during plant growth and leaf senescence. Our results identify NO, as affected by leaf Ca²⁺ level, to be an important negative regulator of leaf senescence initiation. Ca²⁺-mediated NO production during leaf development could control senescence-associated gene (SAG) expression and the production of molecules (such as SA and H₂O₂) that act as signals during the initiation of leaf senescence programs.     

细胞信号转导中Ca2+和微管骨架的关系

就钙离子和微管骨架在信号通路中的关系和二者可能作用方式的研究进展作了概述。     

Plant Innate Immune Response: Qualitative and Quantitative Resistance

Plant diseases, caused by microbes, threaten world food, feed, and bioproduct security. Plant resistance has not been effectively deployed to improve resistance in plants for lack of understanding of biochemical mechanisms and genetic bedrock of resistance. With the advent of genome sequencing, the forward and reverse genetic approaches have enabled deciphering the riddle of resistance. Invading pathogens produce elicitors and effectors that are recognized by the host membrane-localized receptors, which in turn induce a cascade of downstream regulatory and resistance metabolite and protein biosynthetic genes (R) to produce resistance metabolites and proteins, which reduce pathogen advancement through their antimicrobial and cell wall enforcement properties. The resistance in plants to pathogen attack is expressed as reduced susceptibility, ranging from high susceptibility to hypersensitive response, the shades of gray. The hypersensitive response or cell death is considered as qualitative resistance, while the remainder of the reduced susceptibility is considered as quantitative resistance. The resistance is due to additive effects of several resistance metabolites and proteins, which are produced through a network of several hierarchies of plant R genes. Plants recognize the pathogen elicitors or receptors and then induce downstream genes to eventually produce resistance metabolites and proteins that suppress the pathogen advancement in plant. These resistance genes (R), against qualitative and quantitative resistance, can be identified in germplasm collections and replaced in commercial cultivars, if nonfunctional, based on genome editing to improve plant resistance.     

钙与植物乙烯反应的关系研究

研究了Ca2+ 对番茄(Lycopersicon esculentum Mill cv. Lichun)黄化幼苗乙烯反应的影响.通过测定不同Ca2+ 浓度条件下番茄黄化幼苗的"三重反应"、内源乙烯释放量、乙烯受体基因NEVER-RIPE(NR)表达量及胞内CaM含量的变化,结果发现,随着培养基中Ca2+ 浓度从0 mmol/L增加到3.8 mmol/L,番茄黄化幼苗的"三重反应"表型明显增强,内源乙烯释放量、NR基因的表达量及胞内CaM的含量都有不同程度的增加;当Ca2+ 浓度由3.8 mmol/L进一步增加到10 mmol/L时,番茄黄化幼苗"三重反应"表型受到抑制,内源乙烯释放量、 NR基因的表达量及胞内CaM的含量都有所下降.因此,Ca2+ 对番茄黄化幼苗"三重反应"的影响与Ca2+ 调节内源乙烯合成和乙烯受体基因的表达有关,而且Ca2+ 可能是通过CaM含量的变化来调节乙烯作用的.     

非生物逆境胁迫下植物钙信号转导的分子机制

Ca2+作为植物细胞中最重要的第二信使, 参与植物对许多逆境信号的转导。在非生物逆境条件下, 植物细胞质内的钙离子在时间、空间及浓度上会出现特异性变化, 即诱发产生钙信号。钙信号再通过其下游的钙结合蛋白进行感受和转导, 进而在细胞内引起一系列的生物化学反应以适应或抵制各种逆境胁迫。目前在植物细胞中发现Ca2+/CDPK、Ca2+/CaM和Ca2+/CBL 3类钙信号系统, 研究表明它们与非生物逆境胁迫信号转导密切相关。本文通过从植物在非生物逆境条件下钙信号的感受、转导到产生适应性和抗性等方面, 介绍钙信号转导分子机制的一些研究进展。     

非生物逆境胁迫下植物钙信号转导的分子机制

Ca^2＋作为植物细胞中最重要的第二信使,参与植物对许多逆境信号的转导。在非生物逆境条件下,植物细胞质内的钙离子在时间、空间及浓度上会出现特异性变化,即诱发产生钙信号。钙信号再通过其下游的钙结合蛋白进行感受和转导,进而在细胞内引起一系列的生物化学反应以适应或抵制各种逆境胁迫。目前在植物细胞中发现Ca^2＋／CDPK、Ca^2＋／CaM和Ca^2＋／CBL3类钙信号系统,研究表明它们与非生物逆境胁迫信号转导密切相关。本文通过从植物在非生物逆境条件下钙信号的感受、转导到产生适应性和抗性等方面,介绍钙信号转导分子机制的一些研究进展。     

Involvement of Cyclic AMP in ABA- and Ca2--Mediated Signal Transduction of Stomatal Regulation in Vicia faba

The focus of this study is to investigate possible involvementof cyclic AMP in regulation of Vicia stomatal movements. Thepresence of 0.1 mM 8-Br-cAMP, a membrane-permeable analogueof cAMP, alone in the incubation medium did not affect stomatalopening in the light in leaf epidermal peel experiments. However,addition of 0.1 mM 8-Br-cAMP completely reversed exogenous ABA-and C^a2+-induced inhibition of stomatal opening. Consistentwith these results, patch-clamping experiments showed that intracellularaddition of 0.5 mM or 1 mM cAMP significantly reversed the inhibitionof whole-cell inward K⁺ currents by internally supplied 13 µMCa²⁺ or 10 µM ABA in stomatal guard cell protoplasts,respectively. Furthermore, intracellular addition of either10 µM prostaglandin E₁ (PGE1, an adenylate cyclase activator)or 1 mM 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesteraseinhibitor) mimicked the effect of exogenous cAMP on the removalof ABA- or Ca²⁺ inhibition of inward K⁺-current. These resultssuggest that a cAMP signaling pathway is involved in signaltransduction in stomatal regulation by interacting with ABAand Ca²⁺ signaling cascades. A hypothetical mechanism by whichcAMP may regulate K⁺ in stomatal guard cells is also discussed. (Received May 6, 1999; Accepted August 27, 1999)     

Ca2+参与水杨酸诱导蚕豆气孔运动时的信号转导

在一定条件下,外源水杨酸(SA)可以诱导蚕豆(Vicia faba L.)气孔关闭,阻止气孔张开。以Fluo-3—AM作为Ca^2 的荧光探针,利用激光共聚焦扫描显微技术,对水杨酸调控气孔运动中保卫细胞胞质Ca^2 的变化趋势及Ca^2 的来源进行了研究。结果表明,水杨酸可引起胞质Ca^2 增加,这种变化发生在气孔开度改变之前。Ca^2 螯合剂BAPTA（1,2-bis(2-amino phenox-y)ethane-N,N,N′,N′-tetraacetic acid,1mmol/L)几乎可以完全抑制水杨酸诱导气孔开度减小的作用;胞外Ca^2 螯合剂EGTA(2mmol/L)、质膜Ca^2 通道抑制剂尼群地平(nifedipine,NIF,1μmol/L)和LaCl3(1mmol／L)可不同程度地减弱水杨酸诱导气孔关闭的效应。BAPTA(1mmol／L)预处理后,水杨酸不再引起胞质Ca^2 含量改变;尼群地平能够降低水杨酸引起的胞质Ca^2 增加的幅度。说明Ca^2 可能参与水杨酸诱导气孔运动的信号转导。水杨酸引起胞内升高的Ca^2 可能既来自胞外又来自胞内,胞内Ca^2 库可能是其主要来源。     

Activation of Plant Plasma Membrane Ca2+-Permeable Channels by Race-Specific Fungal Elicitors

The response of plant cells to invading pathogens is regulated by fluctuations in cytosolic Ca2+ levels that are mediated by Ca2+-permeable channels located at the plasma membrane of the host cell. The mechanisms by which fungal elicitors can induce Ca2+ uptake by the host cell were examined by the application of conventional patch-clamp techniques. Whole-cell and single-channel experiments on tomato (Lycopersicon esculentum L.) protoplasts revealed a race-specific fungal elicitor-induced activation of a plasma membrane Ca2+-permeable channel. The presence of the fungal elicitor resulted in a greater probability of channel opening. Guanosine 5[prime]-[[beta]-thio]diphosphate, a GDP analog that locks heterotrimeric G-proteins into their inactivated state, abolished the channel activation induced by the fungal elicitor, whereas guanosine 5[prime][[gamma]-thio]triphosphate, a nonhydrolyzable GTP analog that locks heterotrimeric G-proteins into their activated state, produced an effect similar to that observed with the fungal elicitor. Mastoparan, which stimulates GTPase activity, mimicked the effect of GTP[[gamma]]S. The addition of HA1004 (a protein kinase inhibitor) in the presence of the elicitor totally abolished channel activity, whereas okadaic acid (a protein phosphatase inhibitor) moderately enhanced channel activity, suggesting that the activation of the channel by fungal elicitors is modulated by a heterotrimeric G-protein-dependent phosphorylation of the channel protein.     

TRPC5 Is a Ca2+-activated Channel Functionally Coupled to Ca2+-selective Ion Channels

TRPC5 forms non-selective cation channels. Here we studied the role of internal Ca²⁺ in the activation of murine TRPC5 heterologously expressed in human embryonic kidney cells. Cell dialysis with various Ca²⁺ concentrations (Ca²⁺_i) revealed a dose-dependent activation of TRPC5 channels by internal Ca²⁺ with EC₅₀ of 635.1 and 358.2 nm at negative and positive membrane potentials, respectively. Stepwise increases of Ca²⁺_i induced by photolysis of caged Ca²⁺ showed that the Ca²⁺ activation of TRPC5 channels follows a rapid exponential time course with a time constant of 8.6 ± 0.2 ms at Ca²⁺_i below 10 μm, suggesting that the action of internal Ca²⁺ is a primary mechanism in the activation of TRPC5 channels. A second slow activation phase with a time to peak of 1.4 ± 0.1 s was also observed at Ca²⁺_i above 10 μm. In support of a Ca²⁺-activation mechanism, the thapsigargin-induced release of Ca²⁺ from internal stores activated TRPC5 channels transiently, and the subsequent Ca²⁺ entry produced a sustained TRPC5 activation, which in turn supported a long-lasting membrane depolarization. By co-expressing STIM1 plus ORAI1 or the α₁C and β₂ subunits of L-type Ca²⁺ channels, we found that Ca²⁺ entry through either calcium-release-activated-calcium or voltage-dependent Ca²⁺ channels is sufficient for TRPC5 channel activation. The Ca²⁺ entry activated TRPC5 channels under buffering of internal Ca²⁺ with EGTA but not with BAPTA. Our data support the hypothesis that TRPC5 forms Ca²⁺-activated cation channels that are functionally coupled to Ca²⁺-selective ion channels through local Ca²⁺ increases beneath the plasma membrane.     

肝脏固有免疫系统对肠源性感染的免疫应答与免疫耐受机制

肝特殊的解剖结构及生理特征使其成为暴露肠源性抗原的主要器官。由于肝具有独特的固有免疫系统,在正常情况下,肝分布多种致耐受的抗原提呈细胞,对持续性表达或递呈于肝的肠源性抗原物质,诱发针对该抗原的系统性免疫耐受,避免肝受到不必要的免疫损伤。当炎症发生及肝脏固有免疫系统活化时,则通过免疫效应细胞及免疫效应因子对肠源性病原体发挥强烈地免疫应答以控制感染。该过程形成机制的研究对肝功能的理解及肝性疾病的预防与治疗至关重要。本文就肝固有免疫系统对肠源性感染的免疫应答与免疫耐受形成机制作一综述。