| Meprins are oligomeric, cell surface or secreted metalloendopeptidases of the “astacin family”. The studies herein aimed to further elucidate the enzymatic and structural properties of meprin subunits using mutagenesis and molecular modeling. Specifically the active sites of the meprin α and β subunits were investigated to determine residues critical for substrate specificity, and the TRAF (T&barbelow;umor necrosis factor r&barbelow;eceptor-a&barbelow;ssociated f&barbelow;actor) domains were evaluated for their role in oligomerization of the subunits.; Meprins consist of two evolutionarily-related subunits, meprins α and β. The protease domains of meprin α and β are approximately 55% identical at the amino acid level, and their substrate specificity differs markedly. Mutation of meprin αY199 to the amino acid corresponding to the meprin βK185 residue resulted in a protease with more meprin β-like substrate specificity. The meprin α Y199K mutant gained the ability to hydrolyze the β-specific peptide gastrin. The opposite mutation of K185 in the meprin β subunit to Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides demonstrated that meprin residues αY199/βK185 and αF161/βR147 are substrate specificity determinants in meprin subunits. In addition, the combination of the active site investigation and bioinformatics resulted in the identification of new potential substrates and a potentially important new shedding role for meprins.; This work investigated the role of the noncatalytic TRAF domain in meprin multimerization differences. These studies demonstrated the TRAF domain is essential for oligomerization, activation and stability of meprin α. A series of truncation and chimera mutants identified a nine amino acid proposed “oligomerization motif” within the meprin α TRAF domain. This region appears to be critical for multimerization differences between meprin subunits. This work not only addresses the TRAF-mediated associations of meprins, but also has implications for elucidating factors that contribute to homophilic and heterophilic interactions of the entire family of proteins containing TRAF domains. (Abstract shortened by UMI.)... |
