application) N-terminal processing of proteins in eukaryotes occurs both co- and post-translationally and the resulting modifications are involved in translocation, regulation of activity and turnover. The initial reactions involve the removal of the initiator methionine and the addition of N-alpha acetyl groups by methionine aminopeptidases (Met AP) and N-alpha-acetyl transferases (NAT), respectively. Each exists in multiple isoforms that appear to differ in specificity, regulation of cellular location. Using recombinant material, the role of the two types of yeast MetAPs will be determined and the proteins characterized with respect to structural and functional properties, including metal content, substrate specificity and catalytic organization. Site-directed mutagenesis and chemical inhibitors will be extensively used. Cellular localization will also be determined with recombinant fusion proteins and immunological reagents. Recombinant human isoforms will also be prepared and examined for putative glycosylations and the role of the type II enzyme in cell cycle regulation. Similar studies will be carried out with the NATs, with a focus on the subforms specific for N-terminal Met (produced by penultimate Asp, Glu and Asn-the """"""""DEN"""""""" subset). In parallel to the NATs that modify the Gly, Ala, Ser, and Thr (GAST) group, the M-NATs exist as a pair of proteins that may functions as a heterodimer complex. This hypothesis will be tested as a part of the structural characterizations. Two enzymes putatively involved in downstream processing of DEN proteins that will also be investigated. In the first case, yeast will be screened (and enzyme isolated if detected) for an acyl amino acid hydrolase, specific for acetyl Met groups, that could destabilize DEN proteins for degradation by the N-end Rule. In the second, human protein N-terminal asparagine amidohydrolase, which converts N-terminal Asn residues to Asp (the yeast form also acts on Gln), will be isolated and further characterized. Finally, yeast null strains, deficient in several of these (and other) co-/post- translationally active enzymes, will be used to confirm their putative physiological roles and to identify proteins degraded in an N-terminal specific manner.
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