The life expectancy for Americans has been rising continuously. As a result, society faces challenges of age-related diseases. On the other hand, there are still people who suffer untimely death from premature aging. A better understanding of mechanisms underlying aging and age-related diseases is needed to solve these issues. Mice carrying inactivated klotho gene alleles exhibit hallmarks of human aging, suggesting that the Klotho protein is required for the extension of life and the suppression of age-related diseases. The Klotho protein is biosynthesized as a transmembrane protein whose ectodomain is released at the cell surface. The mechanisms for and the biological significance of Klotho ectodomain release are currently unknown. The long-term goal of this study is to identify the protease responsible for generating soluble Klotho and to determine the role of Klotho ectodomain release in suppression of aging and age-related diseases. Preliminary studies have shown that the Klotho ectodomain is released in cultured cells by the tumor necrosis factor-a converting enzyme (TACE). This work will further determine whether TACE is physiologically responsible for Klotho ectodomain release. This question will be addressed by determining the effect of siRNA-mediated TACE expression knockdown on Klotho release in cultured cells, and by comparing the plasma Klotho levels in TACE-null and wild-type mice using an established Klotho radioimmunoassay. The investigators will also identify the TACE cleavage site in transmembrane Klotho by mass spectrometry. They will confirm the cleavage site by constructing a Klotho variant containing mutations at the cleavage site, and determine if the mutations affect Klotho cleavage in cells and with synthetic peptide substrates. Finally, these researchers will investigate the role of TACE-mediated Klotho release in aging by determining whether the activity of Klotho release in vivo correlates with age. This work will reveal insights into the mechanisms that underlie the anti-aging activity of Klotho. It may identify Klotho release as a target for manipulating Klotho activity, which can be used for lifespan extension, and for the prevention and treatment of age-related diseases. [unreadable] [unreadable] [unreadable] [unreadable]