Tumor necrosis factor-a (TNFa) is a cytokine secreted by activated macrophages and monocytes. It plays a central role as a signaling molecule during the induction of inflammatory response, mediating the establishment of a host of acute and chronic inflammatory diseases. It also has a role in human immunodeficiency virus (HIV) activation in acquired immunodeficiency syndrome (AIDS), and in tumor regression in certain types of cancer. The applicant's goal is to study the TNFa converting enzyme (TACE), a zinc metalloprotease of the secretory pathway. Its function is to cleave the membrane-bound precursor TNFa (Pro TNFa) to its mature, soluble form. TNFa is then secreted into the extracellular space, where it exerts its preinflammatory activity. They have recently clones TACE's cDNA, which has substantial homology to the disintegrin metalloproteases. This information, as well as preliminary biochemical characterization, indicates that TACE contains several domains with critical regulatory and mutation function. They propose to elucidate the specific roles of those domains in substrate recognition, maturation/activation and intracellular trafficking of TACE through the following specific aims: 1. The expression and maturation of TACE to its active form requires overcoming negative regulatory signals contained within the TACE polypeptide. They will define what those sequence requirements are for the expression of full-length, active TACE. This will complement the previous characterization of functional, recombinant forms of TACE obtained by overexpression in mammalian and insect cells. In addition, they will investigate the role of the cytoplasmic domain of TACE in negative regulation of the activation and subcellular localization of this protein. This will include the identification of cellular regulatory proteins interacting with it. II. The initial studies suggest that the cysteine-rich (cys) domain of TACE is essential for displacement of the inhibitory pro domain from the catalytic site, resulting in TACE activation. They plan to investigate the role of the cys domain in TACE activation and in proTNFa substrate recognition/binding.
