Peptidases Involved in Signal Peptide Generation and Degradation

Ohio State University Research Foundation -Do Not Use, Columbus OH

Investigators

Abstract

The long-term objective of this project is to understand the structural and functional relationships among the proteases involved in signal peptide generation and degradation in bacteria. Typically, proteins that are targeted to specific subcellular compartments such as the endoplasmic reticulum, mitochondria intermembrane space, or chloroplast thylakoid lumen contain cleavable signal sequences that provide information for export. Signal peptidases are proteases that generate the signal peptide by removing the signal sequence from exported proteins. The signal peptide hydrolases then degrade the signal peptides that can be toxic to the cell or interfere with protein export. The objective of this research is to understand which region of the signal peptidase catalytic domain interacts with the membrane in order to understand how the enzyme cleaves presecretory proteins at the membrane surface. This project also examines the role of signal peptide peptidase A, another novel peptidase, that carries out catalysis by an unconventional mechanism. The project will 1) define the structural elements within signal peptidase that allow it to cleave preproteins with exquisite accuracy, 2) determine the role of the signal peptidase hydrophobic antiparallel Beta-strand below the active site in membrane association, and 3) elucidate the catalytic mechanism, membrane topology, and substrate specificity of signal peptide peptidase A. Site-directed mutagenesis, chemical modification, genetics and biophysics will be used to achieve these aims. The research will determine how much of the catalytic domain of signal peptidase is within the lipid phase of the bilayer. The broader impact of the project is that it will contribute to the understanding of the mechanism of the unconventional serine proteases and help elucidate how catalysis occurs within or at the membrane surface. A high priority will be the training of both undergraduate and graduate students.