G蛋白亚基在味觉信号传导和味蕾细胞更新中的作用

Taste is one of the five special senses humans possess. Taste malfunction can result in many diseases. Full understanding of the taste system can provide new means to preventing and treating taste disorders and other diseases. Recent studies on sweet,savory (umami)and bitter taste receptors and signal transduciton pathways have made some progress. However, it is still an open question about whether fatty taste is a sixth taste, and also, there are some key signaling molecules to be determined and validated, and their effects on taste stem cell regeneration to be defined.The principal investigator of this project has many years of experience in the molecular biology of taste. He has developed a single taste bud cell transcriptome analysis method and identified several important taste signaling molecules, including the sweet and umami receptor component T1R3, G protein subunits Gbeta1,Gbeta3 and Ggamma13. Some in vitro data indicate that Ggamma13 forms a heterotrimeric G protein with alpha-gustducin and Gbeta1 or Gbeta3, mediating sweet, umami and bitter taste sensation, but in vivo data are yet to be obtained. It has been known to be very dificult to nullify Ggamma13’s gene Gng13 using conventional gene knockout approaches because Gng13 is also expressed in other vital organs at early developmental stages. In this project, the PI has generated a knockin mouse line Gng13-flox that has two loxP sites flanking Gng13 exons. By crossing this line with another one expressing Cre in the olfactory sensory neurons, the PI has successfully abolished Ggamma13 expression in these neurons in adult mice, leading to anosmia of the mutant mice. In this project, the researchers will use another mouse line expressing CreErt2 in taste stem cells under the promoter of the Sox2 gene that encodes an important transcription factor in stem cells. Unlike the original Cre protein, CreErt2 has recombinase activity only when bound with the compound tamoxifen. By administering tamoxifen to the mice, the researchers can knock out Gng13 in taste stem cells as well as mature taste bud cells derived from those stem cells. The researchers will monitor the reduction of Ggamma13 proteins in taste buds, and examine any changes in taste bud cells and in responses of the mice to some tastants, and determine the time needed to completely eliminate Ggamma13 proteins in taste buds.The researchers then will comprehensively assess the responses of the mutant mice to various tastants electrophysiologically and behaviorally, examine taste bud cell type compositions and determine taste bud cell turnover rates, prepare taste bud RNA samples and deep sequence the entire transcriptomes. By comparing these sequenced transcriptomes from the knockout samples versus wild-type control, the researchers will identify up-/down- regulated genes that are controlled by taste signal transduction or involved in taste stem cell proliferation and differentiation. Results from this study will contribute to the molecular biology of taste as well as stem cell research.

味觉是人类的五大感觉系统之一。味觉功能直接影响到人们的生活品质，进食失衡和过度都会导致疾病。对味觉系统的深入研究能为预防和治疗有关疾病提供新途径。最近几年,虽然对甜味、鲜味和苦味的受体和信号转导的研究有了进展，但是还有几个重要的信号传导蛋白没有确定，信号传导与味蕾干细胞再生的关系也不清楚。申请人长期从事味觉分子生物学的研究,发现了多个重要的味觉信号传导蛋白，并对味蕾细胞的更新有详细研究。在本项目中,课题组将利用申请人最近构建产生的可用作有条件基因敲除的小鼠，在味蕾干细胞内诱导敲除G蛋白亚基Ggamma13，结合免疫组化、电生理和行为测试、脉冲追踪、味蕾转录本深度测序和生物信息学分析，在体内鉴定Ggamma13在甜味、鲜味、苦味和可能的脂肪味感知中的作用，同时揭示信号传导对味蕾细胞基因表达的调节及对味蕾干细胞再生的影响。研究成果将推进味觉科学和干细胞科学的研究并为临床应用提供新的科学依据。

味觉和嗅觉是二大化学感觉。最近的研究发现味觉受体和嗅觉受体不仅表达在口腔的味蕾和鼻腔的粘膜上，而且也表达在许多口腔外和鼻腔外的组织器官里，很可能发挥着味觉嗅觉之外的有待揭示的其他关键生理功能。G蛋白亚基Gγ13最早是从小鼠味蕾细胞里分离到的，体外的生物化学实验结果显示它是苦味信号通路的重要组成部分，但体内实验证据一直缺乏，因为全身性敲除Gγ13的基因Gng13后小鼠很难成活。我们通过繁殖条件性敲除Gng13的小鼠，成功地敲除了Gng13在味蕾中的表达，双瓶喜好和记舔机测试实验发现敲除小鼠对甜味和鲜味特别是苦味的灵敏度降低，但对脂肪味、酸味和咸味的感知没有变化，证明Gγ13是甜味、鲜味和苦味的重要信号传导蛋白。另外，味蕾是味觉最外围的信息处理器官，5-羟色胺（5-HT）是味蕾内的重要神经递质，我们的研究阐明了5-HT在味蕾内的生物合成过程，有助于理解各种生理病理条件下味觉变化的机理。我们还发现Gγ13、苦味受体等在肠道和犁鼻器中的表达，它们对检测寄生虫入侵、肠炎的发生及犁鼻器感知信息素有着重要作用。Gng13敲除没有改变味蕾内各类细胞数量，对味蕾细胞的基因表达分析发现多个可能与味蕾干细胞分裂分化有关的基因，其中之一是神经胶质细胞粘连分子（NrCAM）。NrCAM不仅在味蕾细胞中表达，也在嗅觉上皮的静默干细胞HBC中表达。NrCAM在嗅觉上皮受损伤时作用到HBC细胞上的EGFR，促使HBC从静默状态进入活跃状态，触发细胞分裂分化，修复嗅觉上皮，恢复嗅觉功能。深入研究味觉信号传导通路在味蕾及体内其它组织器官中的功能将揭示化学感觉受体及其信号蛋白对各器官生理作用的新机制，将推动干细胞生物学的发展，有助于开发新的生物医学治疗途径。