After folding in the endoplasmic reticulum (ER), several proteases sequentially transit through a proton gradient maintained by series of distinct cellular compartments of the secretory pathway en route to their final destination. During this transit, proteases regulate their activation by recognizing the compartment-specific proton concentration by a poorly understood mechanism. The goal of this project is to advance understanding of this mechanism using pro-protein convertases, a family of eukaryotic serine proteases. Pro-protein convertases sense the compartment-specific proton gradient through histidine pH sensors encoded within N-terminal intramolecular chaperone domains. This project will employ biochemical, structural, computational and cell biological approaches to (i) identify the structural determinants of these pH sensors, (ii) establish the mechanism by which the pH sensor functions in pro-protein convertases, and (iii) understand how sequence variations allow for differential regulation of pro-protein convertases. The results from experiments described in this project will provide insights into the general mechanisms by which the compartment-specific proton gradient regulates biological functions of eukaryotic proteins.
Broader impacts: This CAREER development project will provide a unique educational opportunity for undergraduate and graduate students, particularly those from underrepresented minorities, to actively participate in a research project that integrates biophysics, biochemistry, computational and cell biology. New coursework will be created to train students on how to quantify and establish mechanisms for contemporary issues in biological sciences using multidisciplinary approaches. The participants will synergistically join an educational outreach effort in the department to bring current scientific investigation to local middle and high schools. Through summer laboratory employment, high school students will gain research experience and scientific confidence, while they play integral roles in the development of modern technologies. These activities will enhance Oregon Health and Science University's efforts towards the recruitment and retention of underrepresented students and encourage diversity for future applicants.
Intellectual Merits: Proprotein convertases (PCs), which include furin and PC1/3 along with five other paralogs, represent zymogens whose propeptides function as chaperones and potent inhibitors. Before furin and PC1/3 can function as proteases, their inhibitory propeptides must be removed in a precise process of activation that is controlled by organellar pH. Individual PCs undergo activation at unique organellar pH. For example, pro-PC1 undergoes primary cleavage in the endoplasmic reticulum (ER), but unlike pro-furin, it activates at pH 5.5 in the dense core secretory granules (DCSGs). We have established mechanisms by which individual PCs undergo organelle specific, pH-dependent activation. We demonstrated that a conserved histidine (His69) in the furin-propeptide controls the spatiotemporal activation of mature-furin by acting as a sensor. Using biophysical, biochemical, computational and cell based approaches, we showed that in the pH of the TGN, His69 protonation destabilizes the propeptide and exposes a buried cleavage site for proteolysis. Upon cleavage, the propeptide fragments dissociate from furin to release inhibition. Substituting His69 with a leucine removes the pH sensor and blocks precursor activation in the TGN although the protein folds correctly. Since sequence comparisons establish that the residue corresponding to His69 in the furin propeptide is conserved within all PCs, we hypothesized that along with a primary pH sensor additional determinants likely augment the pH-sensitivity to enable differences in pH-dependent activation of individual PC paralogs. Our analyses of propeptides and proteases from the subtilisin family demonstrated that individual proteases contain varying amounts of histidine residues distributed only within propeptides, but not cognate proteases. Moreover, the histidine bias exists only in propeptides of secreted eukaryotic proteases, but not in prokaryotic orthologs. No other amino acids depict a similar bias. Since eukaryotic cells maintain strict control over protein secretion, in part by using the tightly controlled pH gradient maintained in the secretory pathway, we postulated that eukaryotic proteins evolved to enrich histidines within their propeptides to exploit this gradient, thereby regulating activation within specific organelles. On this basis we predicted and subsequently demonstrated that the propeptides of furin and PC1 contain sufficient information for compartment-specific, pH-dependent activation of cognate proteases. We further showed that the pH-dependent properties of the furin propeptide define an activation window for mature furin, which is likely controlled through local conformational changes in its propeptide. While our results demonstrate that His69 is a pH sensor in pro-furin, and the PC1 propeptide encodes information that facilitates pH-dependent activation of mature PC1, they neither define the pH-sensor in the PC propeptide, nor explain how propeptides can differentiate between pH of the TGN (pH~6.5) and DCSGs (pH ~5.5), respectively. Through additional computational and experimental data on propeptides of eukaryotic proteases, we showed that the PC1 propeptide senses pH through a conserved sensor in a manner similar to the furin propeptide, but because it lacks the secondary pH sensor present in the propeptide of furin, it requires a more acidic pH to unfold and undergo auto-proteolysis. Our laboratory also showed which specific histidine in the propeptide of PC1 functions as a pH sensor that regulates the compartment specific activation pro-PC1. Ours results partially explain how propeptides of furin and PC1 display subtle differences in pH dependent activation of cognate proteases. Broader Impact: During the Career Award, the PI mentored 27 students from Benjamin Franklin high school, an ethnically and socio-economically diverse school in proximity to OHSU. In addition, the PI mentored five undergraduate, and four graduate students. High school students were selected on the basis of surveys that probed their perceptions of science and scientific research, opinions and interests about scientific careers, and by personal interviews conducted with assistance from our administrative Director and Graduate students. Selected students received a stipend, and the goals of the internship were to (i)Teach students important skills and attitudes, and expose them to the concept that scientific research and medicine are intimately related, and perhaps open them up to new educational and career possibilities; (ii) improve quantitative literacy and interdisciplinary connectivity in high school students, (iii) help students gain understanding and respect for the process of data-driven investigation, and the way quantitative data can be used to attempt informed decision-making, (iv) to enhance minority student enrollment in scientific research at OHSU, (v) provide a way for global thinking and make them scientifically astute citizens. Students were taught with assistance of graduate students, who obtained ‘hands-on’ experience in the art of mentoring. An active component of this training was to relate theory they learn from their High School Physics and Chemistry classes with contemporary laboratory techniques, Selected students continued through summer, and presented work in Oregon and Washington State Science Fairs and/or in the Intel- and Siemens-Science Competitions. The PIs lab also helped Benjamin Franklin science teachers to conduct experiments, involving DNA isolation, PCR, gel electrophoresis and chromatography. This helped 50 AP- students experience contemporary biological research.
