Regulation of ER Stress Protein Gene Expression

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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. ***