QUORUM SENSING IN HUMAN CELLS

我们实验室以前发现的RTS基因产生一种天然存在的针对人类胸苷合成酶(TS)RNA的反义RNA(RTSalpha)和两种蛋白质(rTSalpha和rTSbeta)。自从最初发现RTS基因产生蛋白质产物和反义RNA以来，该实验室一直在努力鉴定这些蛋白质的生物学功能。在革兰氏阴性细菌中，当细菌在一种称为群体感应的过程中达到高细胞密度时，许多功能(例如生物发光)被激活。群体感应依赖于蛋氨酸代谢产生的物种特异性非极性信号分子(酰基高丝氨酸内酯，acyl-HSLs)的分泌和感知。越来越明显的是，这一过程可能是一种通用的通讯机制，用于调节各种随细胞密度变化的微生物基因功能。本申请中的初步数据提供了证据，证明rTSbeta过表达的细胞以细胞密度依赖的方式调节TS水平。与不过度产生rTSbeta的细胞相比，这种调节是由细胞间信号分子的过度产生控制的。数据进一步表明，这些分子是蛋氨酸的新型非极性代谢物，很可能在化学结构上与微生物群体感应分子相似。我们假设RTS是一种生长抑制基因，通过一种新发现的信号通路来调节细胞生长，这种基因通过一种新发现的信号通路来调节TS蛋白和RNA的下降，这是本应用的主题。由于TS是药物设计和癌症化疗的主要靶点，而rTSbeta在人体肠道组织中表达，这一途径可能是药物开发和调节肿瘤治疗反应的最佳候选途径。我们建议从三个特定的目的来研究这些可能性：1)研究rTSbeta过表达细胞下调反式TS的机制。其他细胞密度调节基因的鉴定。2)检测rTSbeta过表达与蛋氨酸代谢的关系。信号分子的分离与表征。3)建立了酰基HSL的体外合成方法。RTS蛋白和其他参与者在这一反应中的作用的资格。RTS酶的纯化及酰基-HSL生物合成反应的动力学分析。

The rTS gene, previously discovered in our laboratory, produces a naturally occurring antisense RNA (rTSalpha) to human thymidylate synthase (TS) RNAs and two proteins (rTSalpha and rTSbeta). Since the initial discovery that the rTS gene produces protein products as well as antisense RNA, this laboratory has endeavored to identify the biologic functions of the proteins. In gram- negative bacteria, many functions (e.g. bioluminescence) are activated when the bacteria reach high cell densities in a process called quorum sensing. Quorum sensing depends upon the secretion and sensing of species-specific nonpolar signal molecules (acyl homoserine lactones, acyl-HSLs) that arise from the metabolism of methionine. It is becoming evident that this process may be a universal communication mechanism for regulating a variety of microbial gene functions that vary with cell density. Preliminary data in this application provide evidence that rTSbeta over-expressing cells regulate TS levels in a cell density dependent manner. This regulation is controlled by the over-production of intercellular signal molecules compared with cells that do not over-produce rTSbeta. Data further suggest these molecules are novel nonpolar metabolites of methionine, likely to be similar in chemical structure to microbial quorum sensing molecules. We hypothesize the rTS is a growth suppressing gene that regulates cell growth by instigating the decline of TS protein and RNA through a newly discovered signalling pathway that is the subject of this application. Since TS is a major target for drug design and cancer chemotherapy, and rTSbeta is expressed in tissues of the human gut, this pathway may be a prime candidate for drug development and for modulating tumor response to therapy. We propose to investigate these possibilities in three Specific Aims: 1) Study the mechanism for down-regulation of TS in trans by rTSbeta over- expressing cells. Identification of other cell population density regulated genes. 2) Examine the relation of rTSbeta over-expression to Met metabolism. Signal molecule isolation and characterization. 3) Develop an in vitro assay for acyl-HSL synthesis. Qualification of the role of rTS proteins and other participants in this reaction. Purification of rTS enzyme and kinetic analysis of the acyl-HSL biosynthetic reaction.