Regulation of ER Stress Protein Gene Expression

9317238绿色细胞控制膜合成和组装的能力在其执行特定功能或适应不断变化的外部环境的效率中起着关键作用。该建议特别关注内质网（ER）成分的合成和组装的调节，以响应细胞分泌负荷的增加，以及内质网应激蛋白在这一途径中的作用。已经建立了一个实验系统，可以用来探索内质网的“信号传导”，并可以作为研究这种“信号”产生和转导的分子机制的更精细系统的基础。此外，还发现了一种新的内质网应激蛋白，并分析了控制其表达的作用元件。这项工作导致了报告结构的发展，它代表了内质网中产生的调节信号的最终目标。这些数据为研究内质网应激反应调控的两个方面提供了良好的基础：1)作为应激信号最终目标的顺式调控元件和反式调控因子；2)负责信号从内质网转导到细胞核的分子相互作用。在本项目中，实验将旨在识别和表征ERp72基因的反式作用调控元件，ERp72基因是内质网中产生信号的靶标，并确定内质网中信号产生和内质网膜信号转导的机制。在早期的工作中，已经确定了负责基础转录和应激信号刺激转录的5'上游序列。DNA酶I足迹和迁移转移DNA结合研究的结合将用于扩展这些结果，并确定识别这些元素的反式作用因子。这些元素的鉴定将使我们能够完成它们的纯化和克隆，反过来，应该为我们提供用于转导内质网信号的调节途径的有价值的新见解。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抑制剂来探测内质网应激蛋白调节途径，以及纯化和克隆的反式作用因子的功能分析。每个特定的目标都是为了独立地产生重要的新信息。这是我们的希望，每个目标将增强另一个产生内质网应激反应所使用的细胞内信号途径的详细定义。为了使有机体生存，它的细胞必须能够适应自身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它们作出反应。该项目关注的是细胞识别其分泌蛋白数量增加的能力，并通过增加适当处理分泌蛋白所需的细胞机制的数量来应对这种能力（细胞对分泌负荷增加的应激反应）。真核细胞被有效地划分为多个功能区室，其中许多是由细胞膜划分的。这些膜封闭的区室之一，内质网（ER），是新合成的蛋白质最初被放置的地方，如果它们注定要分泌。新的分泌蛋白在内质网内经历一些翻译后加工，然后在它们最终从细胞中分泌出来之前被转运到其他细胞内隔室。当进入内质网的分泌蛋白的数量增加超过系统处理负荷的能力时，一个“信号”被传递到细胞核，以上调某些内质网驻留蛋白的产生，这些蛋白参与了短暂性分泌蛋白的适当“处理”。这个项目的重点是这些常驻蛋白的合成如何成为分泌负荷上调的结果。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地利用真核细胞作为大规模生产感兴趣的特定蛋白质的生物反应器至关重要，因此该项目与生物技术直接相关。其次，更好地理解不同的功能区是如何相互沟通的，以及如何与外部环境沟通的，对于理解生物体如何适应不断变化的环境是至关重要的。＊＊＊

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. ***