Posttranslational proteolysis is a common mechanism required for the synthesis of biologically active proteins and peptides in all eukaryotes examined, including yeast, invertebrates, and mammals. One of the early events in precursor protein maturation is endoproteolysis at the carboxyl side of pairs of basic amino acids (especially-LysArg-and-ArgArg-). This type of endoproteolytic cleavage was initially inferred from the sequences of several endocrine and neuroendocrine precursor proteins. Subsequent studies have revealed a broad spectrum of precursor proteins that require endoproteolysis at pairs of basic amino acids to yield mature peptides, including serum factors, viral polyproteins, growth factors, and receptors. Several activities capable of cleaving at single or paired basic residues in vitro have been proposed as candidates for authentic mammalian precursor processing endoproteases. However, none of these candidate activities have been shown to be a bona fide precursor cleaving endoprotease in vivo. In contrast, genetic and biochemical studies unequivocally identified the gene, KEX2, which encodes the endoprotease required for excision of the peptide mating hormone (alpha-factor) from its precursor in Saccharomyces cerevisiae (baker's yeast) by cleaving the precursor on the carboxyl side of pairs of basic residues (-LysArg- and- ArgArg-). We have recently demonstrated that the human fur gene encodes a KEX2-like, Golgi-associated, calcium-dependent serine-endoprotease, furin, that can efficiently process pro-beta-nerve growth factor in the secretory pathway of mammalian cells. The goal of this proposal is to determine the processes which modulate activation, targeting, substrate specificity, and ultimately the role of the human furin endoprotease in precursor protein processing. First, the substrate specificity of the human furin endoprotease will be identified by studies in vivo. This will be done by co-expressing furin with substrate molecules containing selected mutations at, or near the cleavage site. Second, the furin enzymic activity will be purified and characterized in vitro. The cleavage site specificity and the relative affinity of the furin endoprotease for purified """"""""non-endocrine"""""""" versus endocrine precursor proteins will be evaluated. Third, maturation of the profurin zymogen will be studied. Knowledge gained regarding the cleavage site specificity of the mature protease will be used to study the regulation of this possible autocatalytic maturation step of profurin. Fourth, the requirement for Golgi-localization on the efficiency of furin- directed processing will be examined. Specific emphasis will be on understanding the role of the furin cytoplasmic tail in Golgi localization. Finally, antisense experiments designed to selectively deplete furin activity in vivo will be performed to address the role of furin cultured cells and, ultimately, whether furin is the pro-beta-NFG endoprotease in the hippocampus.
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