灵长类苦味受体基因研究进展

在鲜味、甜味、苦味、咸味和酸味5种味觉形式中,苦味能避免动物摄入有毒有害物质,在动物的生存中发挥着特别重要的作用。苦味味觉的产生依赖于苦味物质与苦味受体的相互作用。苦味受体由苦味受体基因Tas2rs编码,此类基因在不同物种中数量变化较大以适应不同的需求。目前的研究在灵长类中鉴别出了若干苦味受体的配体,并发现有的苦味受体基因所经受的选择压在类群之间、基因之间甚至同一基因不同功能区之间都存在着变化。本文从苦味受体作用的多样性特点,受体与配体的对应关系、受体基因进化模式与食性之间的关系、苦味受体基因的适应性进化方面对灵长类苦味受体基因进行了综述,以期为苦味受体基因在灵长类中的深入研究提供参考。     

猪獾苦味受体T2R2基因的分子克隆与进化分析

苦味的感知是机体有效的自我保护机制之一。文章采用PCR和克隆测序方法首次从猪獾基因组中获得一全长为1 169 bp的苦味受体T2R2基因DNA序列(GenBank登录号: FJ812727)。该序列含有完整的1个外显子(无内含子), 大小为915 bp, 编码304个氨基酸残基。其蛋白质等电点为9.76, 分子量为34.74 kDa。拓扑结构预测显示猪獾T2R2蛋白上含有N-糖基化位点、N-肉豆蔻酰化位点各1个, 蛋白激酶C磷酸化位点2个。整个蛋白质多肽链含有7个跨膜螺旋区, 4个细胞外区和4个细胞内区。亲水性/疏水性分析表明, 猪獾T2R2蛋白质为一疏水性蛋白, 其亲水性区段所占比例较小。种间相似性比较显示, 猪獾T2R2基因与犬、猫、牛、马、黑猩猩和小鼠的T2R2基因cDNA序列相似性分别为91.4%、90.6%、84.4%、85.4%、83.8%、72.1%, 氨基酸序列相似性分别为85.5%、85.8%、74.0%、77.6%、75.3%、61.5%。核苷酸替换计算和选择性检验结果表明, 猪獾T2R2基因与犬、猫、牛、马、黑猩猩和小鼠间存在着强烈的纯净化选择(Purifying selection), 即强烈的功能束缚(Functional constraint), 进一步分析发现该选择作用实际上主要存在于跨膜区。猪獾、犬、猫、牛、马、黑猩猩和小鼠的T2R2基因外显子核苷酸序列构建的基因树与其物种树的拓扑结构是相一致的, 表明T2R2基因适合于构建不同物种间的系统进化树。     

大熊猫苦味受体T2R2基因的克隆与序列分析

Based on bitter taste receptor T2R2 gene sequence of domesticated dog(AB249685), one pair of primers were designed and used to amplify an approximately 1.1 kb DNA fragment from genomic DNA sample of giant panda by using PCR. The PCR products were ligated into the pMD-18T vector, and then transformed into competent cells of E.coli DH5α. The identified positive clone was sequenced. The result showed that the T2R2 gene of giant panda was 1 008 bp in length, and contained complete exon, and 915 bp, encoding 304...     

甜味分子与G蛋白偶联受体Tas1R2/3的相互作用及激活机制

甜味分子与C家族G蛋白偶联受体(G protein-coupled receptor,GPCR)的成员之一甜味受体相互作用,从而激活受体并引起甜味觉的感知。本文简要总结了甜味受体(taste receptor 2 and 3,Tas1R2/3)的结构与功能、甜味分子与受体相互作用并激活受体的机制,并对甜味受体研究领域的发展前景进行了展望。甜味分子与受体相互作用机制的阐明对于理解甜味觉的产生与GPCR的结构与功能具有重要的意义。此外,甜味受体结构与功能的研究可为有针对性地设计新型甜味化合物提供理论基础。     

Wnt信号转导途径及其与结肠癌的关联

Wnt信号转导途径参与细胞多种复杂的生化反应过程。目前认为Wnt通路的组成主要包括：细胞外因子(Wnt)、跨膜受体Frizzled(Frz)、β-连环蛋白(β-catenin)及T细胞趋化因子(Tcell factor,TCF)等一系列蛋白。细胞外因子Wnt激活的信号通过胞质蛋白的相互作用,能够使胞浆内β-连环蛋白保持稳定并累积,β-连环蛋白进而入核与T细胞趋化因子联合作用激活靶基因(多数是参与细胞增殖与凋亡的基因,如：Cyclin D1、c—myc等)的转录,而且在Wnt信号途径的不同阶段有各种蛋白因子进行调控;另外,Wnt胞内信号途径的异常激活与人类各种癌症的产生有联系,尤其是结肠癌变。大多数结肠癌患的转化细胞存在APC(adenomatous polyposis coli)基因缺失突变或失活,导致β-连环蛋白在核内的累积,并能影响相关基因转录,被认为是结肠癌发生的关键因素之一。由于该信号转导途径具有如此重要的作用,因此受到研究越来越多的重视。     

羚牛苦味受体3(Tas2R3)基因的克隆及生物信息学分析

Tas2R3是苦味受体基因家族中一个重要的成员,为了进一步了解和研究羚牛(Budorcas taxicolor)苦味受体基因的结构和功能,本研究对羚牛苦味受体3 (Tas2R3)基因进行了克隆和生物信息学分析(GenBank登录号:MG650195)。结果显示,羚牛Tas2R3基因编码区(coding sequence, CDS)序列全长951 bp,共编码316个氨基酸,以亮氨酸含量最高,谷氨酰胺含量最低。其蛋白质等电点为9.68,分子量为51.96 kD。高级结构功能预测显示,二级结构以α-螺旋为主,蛋白质为碱性、稳定的亲水性蛋白,由4个胞外区、7个跨膜区和4个胞内区组成。预测到2种类型共8个糖基化功能位点和4种类型共15个磷酸化功能位点。通过比较Tas2R3基因种间相似性发现,在偶蹄目中具有很高的同源性,羚牛与绵羊(Ovis aries)的相似性最高(0.98),与褐家鼠(Rattus norvegicus)最低(0.52)。用羚牛、绵羊等12个物种的Tas2R3基因CDS序列构建的NJ树与ME树结构一致,表明Tas2R3基因适合用于构建不同物种间的系统进化树。     

大白菜乙烯受体基因家族分子特征、微同线性与进化分析

采用HMMER与BLAST相结合的方法,在大白菜基因组中挖掘了10个大白菜ERT基因,被命名为BraERT1-10.基因结构分析显示,乙烯受体基因的外显子数目变异大,其范围为1-15.微同线性结果表明,白菜与拟南芥间以及白菜基因组内共有5对基因区段具有较高同线性,每对基因区段间至少共享了8个保守序列模块.蛋白保守结构域和亚细胞定位分析表明,BraERT1、BraERT2和BraERT8都具有N端疏水区域、GAF区、HisKA区、反应调节区,分别被定位于质膜、叶绿体和线粒体;BraERT3、BraERT4和BraERT7虽然有N端疏水区域、GAF区和HisKA区,但没有完整的反应调节区,分别被定位于质膜、质膜和胞质外;其余4个蛋白不具有典型区域,主要定位于核和叶绿体内.进化树结果显示,大白菜ERT基因具有3种类型,分别归属于不同3个类群.本研究为大白菜ERT基因功能研究提供线索,为进一步解析大白菜乙烯信号途径奠定基础.     

细胞质膜受体与信息的跨膜传递

(一)膜受体的概念细胞膜受体是位于细胞膜上的糖蛋白、脂蛋白、糖脂蛋白等,它们能有选择地和细胞外环境中的信号物质相结合,并同时产生效应,使细胞的功能和物质代谢朝着一定方向变化,不同的受体接受不同的化学信号,引起不同的生化变化,一般把受体分成3部分:①分辨部(或鉴别器)是受体分子向着细胞外的部分,能识别外界的化学信号,狭义的受体即指分辨部而言。②转换部(或转换器)将分辨部所接受的信号通过蛋白构象的变化传给效应部。③效应部(或效应器)是向着细胞质的部分,它可引起细胞内部产     

跨膜受体核转位通路的研究进展

跨膜受体可从膜表面进入细胞核内直接调控细胞的生命活动,但其核转位的途径至今尚无定论.已有多种模型分析了跨膜受体的核转位过程,它们均强调受体必须从细胞膜或内吞泡"逃脱"到细胞质后,才能进入细胞核内.然而,内吞.分选-浓缩-膜泡融合-释放模型却诠释了一条不同的跨膜受体核转位通路,这将有利于进一步阐明跨膜受体核转位的模式及其分子机制,并为核靶向药物的开发、目的基因的导入、病毒感染的治疗等应用研究提供新的策略.     

动物与植物线粒体基因组结构的差异—两种进化途径

从进化的角度分析和综合多细胞动物、高等植物、原生动物、藻类和真菌的线粒体基因组的大小和各自的结构特点,并根据作者提出的重复序更与基因结构的起源和进货的模式与途径,得出线粒体通过内共生起源,也是从原始的线粒体基因组向小基因组和大基因组两种方向发展,并有着两种与核基因组的可比较的进货途径的结论。这两种进化途径能很好地说明不同类型的真核生理的线本基因组的结构差异和特点。本文还提出了一个关于重复序列、断列     

Human Bitter Taste Receptors hTAS2R8 and hTAS2R39 with Differential Functions to Recognize Bitter Peptides

The strong bitter peptide, Phe-Phe-Pro-Arg, activated cultured cells expressing either of the known human bitter taste receptors, hTAS2R8 and hTAS2R39. The partial structure of Pro-Arg activated hTAS2R39, but did not activate hTAS2R8. These receptors may not indiscriminately recognize bitter peptides, but have a differential function in their recognition.     

Evolutionary insights into umami,sweet, and bitter taste receptors in amphibians

Umami and sweet sensations provide animals with important dietary information for detecting and consuming nutrients, whereas bitter sensation helps animals avoid potentially toxic or harmful substances. Enormous progress has been made toward animal sweet/umami taste receptor (Tas1r) and bitter taste receptor (Tas2r). However, information about amphibians is mainly scarce. This study attempted to delineate the repertoire of Tas1r/Tas2r genes by searching for currently available genome sequences in 14 amphibian species. This study identified 16 Tas1r1, 9 Tas1r2, and 9 Tas1r3 genes to be intact and another 17 Tas1r genes to be pseudogenes or absent in the 14 amphibians. According to the functional prediction of Tas1r genes, two species have lost sweet sensation and seven species have lost both umami and sweet sensations. Anurans possessed a large number of intact Tas2rs, ranging from 39 to 178. In contrast, caecilians possessed a contractive bitter taste repertoire, ranging from 4 to 19. Phylogenetic and reconciling analysis revealed that the repertoire of amphibian Tas1rs and Tas2rs was shaped by massive gene duplications and losses. No correlation was found between feeding preferences and the evolution of Tas1rs in amphibians. However, the expansion of Tas2rs may help amphibians adapt to both aquatic and terrestrial habitats. Bitter detection may have played an important role in the evolutionary adaptation of vertebrates in the transition from water to land.     

Top-Down Control of Sweet and Bitter Taste in the Mammalian Brain

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Contrasting modes of evolution between vertebrate sweet/umami receptor genes and bitter receptor genes

Taste reception is fundamental to diet selection in many animals. The genetic basis underlying the evolution and diversity of taste reception, however, is not well understood. Recent discoveries of T1R sweet/umami receptor genes and T2R bitter receptor genes in humans and mice provided an opportunity to address this question. Here, we report the identification of 20 putatively functional T1R genes and 167 T2R genes from the genome sequences of nine vertebrates, including three fishes, one amphibian, one bird, and four mammals. Our comparative genomic analysis shows that orthologous T1R sequences are relatively conserved in evolution and that the T1R gene repertoire remains virtually constant in size across most vertebrates, except for the loss of the T1R2 sweet receptor gene in the sweet-insensitive chicken and the absence of all T1R genes in the tongueless western clawed frog. In contrast, orthologous T2R sequences are more variable, and the T2R repertoire diverges tremendously among species, from only three functional genes in the chicken to 49 in the frog. These evolutionary patterns suggest the relative constancy in the number and type of sweet and umami tastants encountered by various vertebrates or low binding specificities of T1Rs but a large variation in the number and type of bitter compounds detected by different species. Although the rate of gene duplication is much lower in T1Rs than in T2Rs, signals of positive selection are detected during the functional divergences of paralogous T1Rs, as was previously found among paralogous T2Rs. Thus, functional divergence and specialization of taste receptors generally occurred via adaptive evolution.     

Taste solution preferences of C57BL/6J and 129X1/SvJ mice: influence of age, sex, and diet

To examine whether age influences taste solution preferences, we measured taste preferences of C57BL/6J and 129X1/SvJ mice given a series of 48-h 2-bottle tests with a choice between water and one of the following taste solutions: 2 mM saccharin, 5 mM citric acid, 30 microM quinine hydrochloride, 75 mM sodium chloride (NaCl), 10 mM inosine monophosphate (IMP), 50 mM calcium chloride (CaCl(2)), and 10% ethanol. We tested separate groups of male mice fed Teklad 8604 chow at ages 4, 6, 9, 12, 15, 20, 25, 30, 40, and 50 weeks and retested some of these mice at 54, 75, and 100 weeks and again at 125 weeks. Female mice fed chow were tested at ages 4, 12, 25, and 50 weeks and retested at 54, 75, 100, and 125 weeks. Male mice fed AIN-93G semisynthetic diet were tested at ages 4, 12, 25, and 50 weeks and retested at 54, 75, and 100 weeks. Concentration-response functions for each taste solution were collected from male and female mice fed chow aged 8 or 125 weeks. In general, the results showed that age had little effect on taste preferences. Exceptions included 1) a small increase in quinine hydrochloride preference between 54 and 125 weeks in mice of both strains and sexes, 2) a marked increase in NaCl preference between 4 and 12 weeks in female B6 mice, 3) a gradual decrease in IMP preference between 4 and 125 weeks in male and female 129 mice, 4) a marked decrease in CaCl(2) preference between 54 and 125 weeks in male and female 129 mice, and 5) a marked reduction in ethanol preference between 4 and 12 weeks in male B6 mice fed AIN-93G diet but not chow. These results show that over a wide range and with the exceptions noted, age contributes little to the variation in taste preferences observed in C57BL/6J and 129X1/SvJ mice.     

Key Amino Acid Residues Involved in Multi-Point Binding Interactions between Brazzein, a Sweet Protein, and the T1R2-T1R3 Human Sweet Receptor

The sweet protein brazzein [recombinant protein with sequence identical with the native protein lacking the N-terminal pyroglutamate (the numbering system used has Asp2 as the N-terminal residue)] activates the human sweet receptor, a heterodimeric G-protein-coupled receptor composed of subunits Taste type 1 Receptor 2 (T1R2) and Taste type 1 Receptor 3 (T1R3). In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: site 1 (Loop43), site 2 (N- and C-termini and adjacent Glu36, Loop33), and site 3 (Loop9-19). Basic residues in site 1 and acidic residues in site 2 were essential for positive responses from each assay. Mutation of Y39A (site 1) greatly reduced positive responses. A bulky side chain at position 54 (site 2), rather than a side chain with hydrogen-bonding potential, was required for positive responses, as was the presence of the native disulfide bond in Loop9-19 (site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus flytrap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in brazzein response. The exception, hT1R2 (human T1R2 subunit of the sweet receptor):R217A/hT1R3 (human T1R3 subunit of the sweet receptor), which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site is involved in subunit-subunit interaction rather than in direct brazzein binding. Results from this study support a multi-point interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models.     

Integration of QSAR modelling and QM/MM analysis to investigate functional food peptides with antihypertensive activity

Antihypertensive peptides derived from dietary proteins have long been recognised as an important source of developing functional foods with blood pressure-lowering effect. However, most of such peptides exhibit diverse tastes, such as sweet, bitter, sour and salty, which is a non-negligible aspect considered in the food development process. In the present study, several predictive quantitative structure–activity relationship (QSAR) models that correlate peptide's structural features with their multi-bioactivities and bitter taste are established at both sequence and structure levels, and the models are then used to conduct extrapolation on thousands of randomly generated, structurally diverse peptides with chain lengths ranging from two to six amino acid residues. Based on the statistical results gained from QSAR modelling, the relationship between the antihypertensive activity and bitter taste of peptides at different sequence lengths is investigated in detail. Moreover, the structural basis, energetic property and biological implication underlying peptide interactions with angiotensin-converting enzyme (ACE), a key target of antihypertensive therapy, are analysed at a complex three-dimensional structure level by using a high-level hybrid quantum mechanics/molecular mechanics scheme. It is found that (a) bitter taste is highly dependent on peptide length, whereas ACE inhibitory potency has only a modest correlation with the length, (b) dipeptides and tripeptides perform a moderate relationship between their ACE inhibition and bitterness, but the relationship could not be observed for those peptides of more than three amino acid residues and (c) the increase in sequence length does not cause peptides to exhibit substantial enhancement of antihypertensive activity; this is particularly significant for longer peptides such as pentapeptides and hexapeptides.     

转铜/锌超氧化物歧化酶和抗坏血酸过氧化物酶基因甘薯的耐旱性

水分胁迫使转铜／锌超氧化物歧化酶基因（Cu／Zn SOD）和抗坏血酸过氧化物酶基因（APX）甘薯及未转基因甘薯中超氧阴离子（O2^-）、过氧化氢（H2O2）、丙二醛（MDA）含量和细胞膜相对透性增加,在相同条件下以上指标均为转基因甘薯低于未转基因甘薯;而叶片含水量、净光合速率（Pn）和气孔导度（Gs）均下降,SOD和APX酶活性随胁迫程度的加重先增大后减小,胞间CO2浓度（Ci）则先减小后增大,在相同条件下转基因甘薯中以上指标均高于未转基因甘薯。这些结果表明：转入Cu／Zn SOD和APX基因使转基因甘薯清除活性氧的能力增强,在水分胁迫下能保持较高的叶片含水量和Pn,耐旱性得到提高。