QUORUM SENSING IN HUMAN CELLS

人体细胞中的群体感应

• 批准号: 6610990
• 负责人: BRUCE JEFFREY DOLNICK
• 金额: $ 23.09万
• 依托单位: ROSWELL PARK CANCER INSTITUTE CORP
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6610990
• 关键词: acyl group; affinity chromatography; aminoacid metabolism; bioassay; biosynthesis; cell growth regulation; chemical kinetics; enzyme activity; enzyme induction /repression; lactones; mass spectrometry; methionine; serine; thymidylate synthase; tissue /cell culture

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.

先前在我们的实验室中发现的RTS基因产生了自然存在的反义RNA（RTSALPHA），可用于人胸苷酸合酶（TS）RNA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以及反义RNA以来，该实验室一直努力鉴定蛋白质的生物学功能。 在革兰氏阴性细菌中，当细菌在称为Quorum Sensing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。 法群传感取决于由蛋氨酸的代谢产生的物种特异性非极性信号分子（酰基同氨酸内酯，酰基-HSLS）的分泌和传感。 显而易见的是，此过程可能是调节随细胞密度变化的各种微生物基因函数的通用通信机制。 本应用程序中的初步数据提供了证据表明，RTSBETA过表达细胞以细胞密度依赖性方式调节TS水平。 与不会过量产生RTSBETA的细胞相比，该调节是由细胞间信号分子过量产生的控制。 数据进一步表明，这些分子是蛋氨酸的新型非极性代谢产物，在化学结构上可能与微生物群体传感分子相似。 我们假设RTS是一种抑制生长基因，通过通过新发现的信号传导途径促进TS蛋白和RNA的下降来调节细胞的生长，这是该应用程序的主题。由于TS是药物设计和癌症化学疗法的主要靶点，并且RTSBETA在人肠道的组织中表达，因此该途径可能是药物发育的主要候选者并调节肿瘤对治疗的反应。 我们建议在三个特定目的中研究这些可能性：1）研究RTSBETA过表达细胞在反式中下调TS的机制。 鉴定其他细胞种群密度调节基因。 2）检查RTSBETA过表达与MED代谢的关系。 信号分子隔离和表征。 3）开发用于酰基-HSL合成的体外测定法。 RTS蛋白和其他参与者在此反应中的作用的资格。 RTS酶的纯化和酰基-HSL生物合成反应的动力学分析。