Regulation of ER Stress Protein Gene Expression

9317238绿色 细胞控制其膜的合成和组装的能力在它们执行专门功能或适应不断变化的外部环境的效率中起着关键作用。 该建议特别关注响应于细胞分泌负荷增加的内质网（ER）组分的合成和组装的调节以及ER应激蛋白在该途径中的作用。 已经建立了一个实验系统，它可以用来探索从ER的“信号”，这可以是更精细的系统，其中研究的分子机制的产生和转导这种“信号”的基础。 此外，还发现了一种新的内质网应激蛋白，并对其表达调控元件进行了分析。 这项工作导致了报告构建体的发展，这些构建体代表了ER中产生的调节信号的最终靶点。 这些数据提供了一个很好的基础，研究两个方面的调节ER应激反应：1）顺式作用的调节元件和反式作用的因素，是最终的目标，为应力信号;和2）分子相互作用负责的信号转导从ER到核。 在这个项目中，实验将被设计为识别和表征ERp72基因的反式作用调控元件，ER中产生的信号的目标，并确定在ER中的信号产生和跨ER膜的信号转导的机制。 负责基础和应激信号刺激转录的5 '上游序列已经在早期工作中被定义。 结合DNA酶I足迹迁移率变化DNA结合研究将被用来扩展这些结果，并确定识别这些元素的反式作用因子。 这些元件的鉴定将使我们能够完成它们的纯化和克隆，反过来，应该为我们提供有价值的新的见解，用于调节ER信号的调节途径。 这些研究的第二个重点将是药理学研究，涉及使用细胞内信号传导抑制剂来探测ER应激蛋白调节途径和纯化和克隆的反式作用因子的功能分析。 每一个具体目标都旨在独立地产生重要的新信息。 我们希望这两个目标能相互补充，对内质网应激反应所使用的细胞内信号通路产生一个详细的定义。 为了使生物体生存，它的细胞必须能够适应环境或外部环境的变化。 这需要感知变化（信号），然后以适当的方式对其做出反应。 本项目的重点是细胞识别其产生的分泌蛋白量增加的能力，并通过增加适当处理分泌蛋白所需的细胞机器的量来对此做出反应（细胞应激反应增加分泌负荷）。 真核细胞被有效地划分为功能区室，其中许多区室由细胞内膜划定。 这些膜封闭的隔间之一，内质网（ER），是新合成的蛋白质最初放置的地方，如果它们注定要分泌。 新的分泌蛋白在ER内经历一些翻译后加工，然后在它们最终从细胞分泌之前穿梭到另外的细胞内隔室。 当进入ER的分泌蛋白的量增加超过系统处理负荷的能力时，"信号"被传递到细胞核以上调某些ER驻留蛋白的产生，所述ER驻留蛋白参与短暂分泌蛋白的适当"处理"。 该项目的重点是这些常驻蛋白质的合成如何作为分泌负荷的结果而被上调的问题。 该项目具有双重意义。 首先，更好地了解细胞如何处理新合成的分泌蛋白对于有效利用真核细胞作为生物反应器大规模生产特定的感兴趣蛋白质至关重要，因此该项目与生物技术直接相关。 其次，更好地了解不同的功能区室如何相互沟通，以及与外部环境是必不可少的生物体如何适应不断变化的环境的一般性理解。 ***

9317238 Green The ability of cells to control the synthesis and assembly of their membranes plays a critical role in the efficiency with which they perform specialized functions or to adapt to a changing external environment. This proposal focuses specifically on the regulation of the synthesis and assembly of components of the endoplasmic reticulum (ER) in response to an increase in the secretory load of the cell and on the role of the ER stress proteins in this pathway. An experimental system has been established which can be used to explore "signaling" from the ER and which can be the basis for more refined systems in which to study the molecular mechanism of the generation and transduction of this "signal". In addition, a new ER stress protein has been identified and th ecis-acting elements that control its expression have been analyzed. This work has led to the development of reporter constructs which represent the ultimate targets of regulatory signals generated in teh ER. These data provide an excellent basis for the investigation of two aspects of the regulation of the ER stress response: 1) the cis-acting regulatory elements and the trans-acting factors that are the ultimate targets for the stress signal; and 2) the molecular interactions responsible for the transduction of the signal from the ER to the nucleus. In this project, experiments will be designed to identify and characterize the trans-acting regulatory elements of the ERp72 gene, a target for the signal generated in the ER, and to determine the mechanisms of both signal generation in the ER and signal transduction across the ER membrane. The 5' upstream sequences responsible for both basal and stress signal stimulated transcription have already been defined in earlier work. A combination of DNase I footprinting an mobility shift DNA binding studies will be used to extend these results and to identify trans-acting factors recognizing these elements. The identification of these el ements will allow us to accomplish their purification and cloning and, in turn, should provide us with valuable new insights into the regulatory pathway used to transduce the ER signal. A second focus of these studies will be pharmacological studies involving the use of inhibitors of intracellular signalling to probe the ER stress protein regulatory pathway and the functional analysis of purified and cloned trans-acting factors. Each of the specific aims is designed to produce significant, new information independently. It is our hope that each aim will augment the other a produce a deteailed definition of the intracellular signal pathway used by the ER stress response. %%% In order for an organism to survive, its cells must be able to adapt to accomodate changes in its circumstances or its external environment. This entails sensing the changes (signals) and then responding to them in an appropriate manner. This project focuses on the ability of a cell to recognize an increase in the amount of secretory proteins it is producing and to respond to this by increasing the amounts of the cellular machinery required to deal with secretory proteins appropriately (cellular stress response to increased secretory load). The eukaryotic cell is efficiently partitioned into functional compartments, many of which are delineated by intracellular membranes. One of these membrane-enclosed compartments, the endoplasmic reticulum (ER), is the place where newly-synthesized proteins are initially placed if they are destined for secretion. The new secretory proteins undergo some post-translational processing within the ER, and are then shuttled to additional intracellular compartments before their ultimate secretion from the cell. When the amount of secretory protein coming into the ER is increased beyond the system's ability to handle the load, a "signal" is transmitted to the nucleus to up-regulate production of certain ER resident proteins which are involved in the prope r "handling" of transitory secretory proteins. This project focuses on the question of how synthesis of those resident proteins become up-regulated as a result of secretory load. The project has two-fold significance. First, a better understanding of how cells handle newly-synthesized secretory proteins is critical to efficient exploitation of eukaryotic cells as bioreactors for the large-scale production of specific proteins of interest, and therefore this project has direct relevance to biotechnology. Second, a better understanding of how different functional compartments communicate with each other as well as with the external environment is essential to a general understanding of how organisms adapt to changing circumstances. ***