The broad, long term objective of this project is to develop therapeutic approaches to block apoptosis. The major inducible heat shock protein Hsp72 protects cells from most stresses and thus represents a generalized repair/stress resistance component. Hsp72 not only refolds damaged proteins but also interferes with the cell death signaling pathway thus providing cells with time to repair the damage. A protein factor (HIF) from human ascites fluid activated Hsp72 synthesis in cell cultures. HIF protected cells from apoptosis caused by heat shock, mutagen treatment, and oxidative damage. Isolation and cloning of this extracellular apoptosis inhibitor is the goal of this study. The specific aims of this study are: 1. We will purify the anti-apoptotic (HIF). A number of chromatographic approaches will be used in a bioassay-driven purification of human HIF. Expression of Hsp72 by fibroblasts will be used as a test The purity of active fractions will be tested by SDS-PAGE and 2-D electrophoresis. 2. We will clone the HIF gene. When HIF is purified, we will use a partial protein sequence to prepare degenerate oligonucleotides to identify the gene in a human genomic library. The clone will be partially sequenced, and a specific probe will be used to screen a cDNA library. The coding region will be completely sequenced, and this will provide useful information regarding the nature of the factor. Significance. Establishing the nature of a systemic apoptotic inhibitor that promotes cell repair will lay the basis for learning how an organism controls the fate of its cells. Relevance to human health. A natural extracellular inhibitor of apoptosis will serve as a basis for developing effective therapies for treating such stress- related conditions as ischemia and neurodegenerative disorders, where apoptosis in response to damage is an important factor in disease. PROPOSED COMMERCIAL APPLICATIONS: The research will lead to the identification of a natural human inhibitor of apoptosis that will be used to develop therapies against a number of diseases, such as ischemia and neurodegenerative disorders
