Decoding intramembrane aspartyl protease substrate preferences and activity

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Cells communicate through chemical signals. The fidelity of signaling in the cell is critical to maintaining overall health and is controlled by signaling molecules that are generated by a variety of enzymes. Of interest here is the category of signaling molecules generated via the action of the breakdown of proteins or peptides, known as proteolysis, performed by intramembrane proteases (IPs), which are enzymes found in the water-depleted lipid membrane of the cell. Although proteolysis performed by proteases is well-known, IPs conduct this water-dependent chemistry in the water-depleted cell membrane. The action of IPs is key to cell division, development, and metabolism, but this unique chemical environment renders the system difficult to study and thus key details of the process remain unknown. The cohesive research/educational mission at the Georgia Institute of Technology involves students at high school, undergraduate, and graduate levels to comprehend, at the molecular level, the chemistry and structure of a particular IP type. The research will answer how the specific IP performs proteolysis chemistry on its many substrates in the distinctive membrane environment to generate the signaling molecule products, as a model for the larger IP family. To answer these questions, students will use cross-disciplinary, cutting edge, and high-resolution biochemical and biophysical methods tailored to membranes, training them for future STEM careers. In turn, educational modules for K-12 students will be developed, implemented, and disseminated. In the long term, an understanding of the chemistry and structure of IPs will lead to future research to better understand how IP-derived signaling is regulated at the cellular, tissue, and organismal level to elicit specific biological outcome.The aspartyl IP sub-class (IAP) will be studied, using as a model an IAP ortholog from the archaeon Methanoculleus marisnigri JR1. Students will probe the interplay between chemical and positional considerations, evaluate the extent of cleavage processivity, and elucidate substrate-IAP interactions in molecular detail, using tailored high-resolution and quantitative experimental methods. Students will then study the structure of activated substrate/IAP complexes using chemical crosslinking, X-ray crystallography, and small angle neutron scattering, results of which will be complemented by molecular dynamics simulations. These studies will provide critical new mechanistic insight into the process of intramembrane proteolysis using a well-defined model system. Results of these studies will challenge tenets in the field based on lower resolution or indirect methods, enable the identification substrates for IAPs broadly throughout biology, and will lead to future studies to probe the mechanisms by which intramembrane proteases are regulated. New structure-based hypotheses for further study, including the origin of bulk water entry for catalysis, will emerge. The tailored quantitative methods also hold promise for broad adoption across IAP orthologs, experimental conditions, and IP classes where similar knowledge is desirable. Any unprecedented strategies used for structure determination will be applicable to studies of other IAPs, other IP classes, and membrane proteins. Results will be broadly disseminated in the cross-disciplines of biochemistry, proteomics, membrane biophysics, structural biology, and neutron sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.