The project will focus on the structure and enzymology of signal peptidase, a membrane-bound proteolytic enzyme that removes amino-terminal signal peptides from nascent secretory proteins as they are transported into the lumen of the endoplasmic reticulum. The mechanism of action of this proteinase has not yet been determined in spite of the central role of this enzyme in the biosynthesis of secreted proteins. The catalytic mechanism of signal peptidase may define a new class of proteolytic enzymes. Mutations in signal peptide sequences that directly affect signal peptidase have been discovered and shown to result in human disease. The nature of the two proteins that constitute hen oviduct microsomal signal peptidase will be examined and their functions defined. The protein(s) minimally required for enzymatic activity will be determined. Synthetic peptide analogs bearing covalently attached photoactivatible cross linking sites will be used to identify the roles of the protein subunits in the catalytic action of signal peptidase. The effect of naturally occurring mutations in the signal peptide regions of two human proteins, coagulation factor X and serum albumin, will be examined to provide a detailed description of the effects of these genetic mutations on the biosynthesis of these proteins. A general approach for studying the effect of mutations of signal peptides on signal peptidase will be developed. A cDNA clone encoding the 19 kDa protein of hen oviduct signal peptidase will be cloned and characterized. An important goal of the cloning of the 19 kDa subunit is to determine whether there are also two distinct genes encoding related hen oviduct signal peptidase proteins of this size, as has been demonstrated in the canine signal peptidase. cDNA clones encoding signal peptidase protein subunits will be expressed in a heterologous cell system to produce enzymatically active protein(s). Successful development of this approach will lead to future studies using site-directed mutagenesis to probe the enzymatic mechanism this proteinase. Completion of these studies will yield a detailed understanding of the structure and function of microsomal signal peptidase which plays a key role in the early phase of the biosynthesis of secreted proteins in eukaryotes.
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