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Lessons Learned from Structural Functional Analyses
University of Puerto Rico
Thesis Advisor: Huizhou Fan, PhD
Graduate Program in Physiology and Integrative Biology
RWJMS Research Tower
Tuesday, July 29, 2008
Tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) is a multidomain zinc metalloprotease that catalyzes ectodomain shedding of numerous membrane-anchored proteins with diverse biological activities in a highly regulated fashion. To understand how TACE activity is controlled we performed structural-functional analyses in the catalytic domain, transmembrane domain (TM) and cytoplasmic domain. In contrast to other metzincin superfamily members that retain enzyme activities when the conserved met-turn methionine is replaced, TACE requires the met-turn Met435 for cleaving transmembrane TNF-alpha, transforming growth factor-alpha (TGF-alpha) and L-selectin. Comparative modeling and dynamic computation support the notion that Met435 is required to maintain the stability of the catalytic center of TACE for the hydrolysis of peptide bonds in substrates. A potential regulatory role for the TM in ectodomain shedding was determined by using a chimeric approach. Interestingly, substitution of the TACE TM with that of the prolactin receptor or platelet-derived growth factor receptor resulted in severe loss of TGF-alpha shedding, but had no effects on the cleavage of TNF-alpha and L-selectin. In addition, a TM-free TACE construct that is anchored to the plasma membrane by a glycosylphosphatidylinositol-binding polypeptide failed to restore shedding of TGF-alpha, TNF-alpha and L-selectin in cells lacking endogenous TACE activity. Therefore, it appears that anchorage of TACE to the lipid bilayer through a TM is required for efficient cleavage of a broad spectrum of substrates, and that the amino-acid sequence of TACE TM may play a role in regulatory specificity among TACE substrates. Finally, substitution of phosphorylated serine and threonine residues with aspartic acid in the cytoplasmic domain of TACE resulted in functional recovery of the shedding-defective C600Y TACE variant. Expression analysis suggests that phosphorylation stimulates TACE maturation. These findings may have important implications for the regulation of TACE-mediated ectodomain shedding in physiology and pathogenesis.