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.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Pathobiochemistry Study Section (PBC)
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Gretz, Elizabeth
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University of Pennsylvania
Schools of Medicine
United States
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Gonzales, Patricia E; Galli, Jennifer D; Milla, Marcos E (2008) Identification of key sequence determinants for the inhibitory function of the prodomain of TACE. Biochemistry 47:9911-9
Sagi, Irit; Milla, Marcos E (2008) Application of structural dynamic approaches provide novel insights into the enzymatic mechanism of the tumor necrosis factor-alpha-converting enzyme. Anal Biochem 372:1-10
Milla, Marcos E; Gonzales, Patricia E; Leonard, Jennifer D (2006) The TACE zymogen: re-examining the role of the cysteine switch. Cell Biochem Biophys 44:342-8
Leonard, Jennifer D; Lin, Frank; Milla, Marcos E (2005) Chaperone-like properties of the prodomain of TNFalpha-converting enzyme (TACE) and the functional role of its cysteine switch. Biochem J 387:797-805
Gonzales, Patricia E; Solomon, Ariel; Miller, Ann B et al. (2004) Inhibition of the tumor necrosis factor-alpha-converting enzyme by its pro domain. J Biol Chem 279:31638-45
Solomon, Ariel; Rosenblum, Gabriel; Gonzales, Patricia E et al. (2004) Pronounced diversity in electronic and chemical properties between the catalytic zinc sites of tumor necrosis factor-alpha-converting enzyme and matrix metalloproteinases despite their high structural similarity. J Biol Chem 279:31646-54
Mohan, Mohita J; Seaton, Theresa; Mitchell, Justin et al. (2002) The tumor necrosis factor-alpha converting enzyme (TACE): a unique metalloproteinase with highly defined substrate selectivity. Biochemistry 41:9462-9
Skovronsky, D M; Fath, S; Lee, V M et al. (2001) Neuronal localization of the TNFalpha converting enzyme (TACE) in brain tissue and its correlation to amyloid plaques. J Neurobiol 49:40-6