Characterizing the Interplay between Dynamics and Regulation in the Trypsinogen/Trypsin Protease System

Abstract

Trypsin (Tn) is a digestive serine protease found in eukaryotes and prokaryotes. Tn is manufactured by the vertebrate pancreas as its proenzyme trypsinogen (Tgn) which includes an N-terminal six amino acid zymogen region. The zymogen form, Tgn, is disabled against cleavage of substrate until Tgn reaches the small intestine. Within the small intestine, enterokinase cleaves the inhibiting peptide from folded Tgn to release active Tn protease. Fluorescence experiments with bovine Tn demonstrate remarkably slow global unfolding, with a kinetic barrier to unfolding as large as kinetically stabilized bacterial homologues. Whereas bacterial homologues have evolved to resist proteolysis, thereby prolonging extracellular activity, mammalian Tgn/Tn is tightly regulated through a series of auto-cleavage events. We propose that Tgn/Tn experiences subglobal dynamics, and that sampling of these semi-unfolded states under physiological conditions causes this rapid autolysis. To explore this, we will perform native-state hydrogen exchange mass spectrometry of a purified inactive mutant. Exploring the Tgn/Tn subglobal dynamics which expose the native-state to autolysis will grant understanding of the link between Tn dynamics and the regulation of its function through its specific sequence of auto-cleavage events. By comprehensively characterizing the sampling of subglobally and globally unfolded states of Tgn/Tn, more can be inferred about the occurrence of non-native state protein aggregation and susceptibility to proteolytic attack. Studying Tgn/Tn from the inactive Tgn/Tn mutant allows direct assessment of the role of subglobal protein dynamics and the regulation of these dynamics in Tn folding disorders without the complication of autoproteolysis. The Tgn/Tn system will allow investigation of diseases characterized by abnormal protein aggregation and vulnerability to proteolytic attack, in addition to diseases directly related to Tn function such as pancreatitis.