Natural and synthetic mechanisms of ligand formation

PROJECT SUMMARY/ABSTRACT Cells tightly regulate secreted signaling proteins so that they function at the right place and time. Most signaling proteins form complexes with other signals in various combinations. This mix-and-match strategy is deployed in all stages of metazoan evolution, ultimately enabling cell type diversity and complex animal behaviors. But what are the molecular rules that govern the formation of signaling ligands? The overarching goal of my research program is to describe the fundamental mechanisms of signal assembly and processing, as well as to provide solutions when signaling goes awry. The first arm of the program investigates transforming growth factor-beta signaling proteins, Vg1 and Nodal, that must assemble as heterodimers to properly induce the mesoderm and endoderm tissues (e.g., muscle, bone, blood). We recently discovered that several chaperones aid in the robust and selective assembly of Vg1-Nodal heterodimers in animal embryos. This finding has opened fundamental mechanistic questions on chaperone-mediated signal assembly: What are the molecular rules (and the order of these rules) that chaperones use to control the composition of signaling complexes? We will combine embryological manipulation, biochemical reconstitution in vitro, and computational modeling to identify the protein regulators and binding motifs that govern the heteromeric assembly of signaling proteins. In the second arm of the program, we aim to assign the true physiological function of endogenous peptides. For example, a single polyprotein-encoding gene can produce up to eight bioactive peptides. However, cells only use a handful of convertases to process the thousands of secreted precursor proteins and peptides. In our previous work, we established a new molecular approach to process secreted proteins, the Synthetic Processing (Synpro) system. The Synpro system is composed of a family of secreted, synthetic proteases that can cleave cognate sequences on any secreted protein. We will further develop these novel secreted proteases to cleave secreted polyproteins in a sequence-specific way. Using the Synpro system, our lab will assign peptide function in two ways: (i) introduction of Synpro-cleavable sequences into polyproteins or (ii) directed evolution of Synpro proteases to process the natural sequence of each peptide within a polyprotein. Diversifying the cleavage sequence alphabet of Synpro proteases will enable us to assign peptide function, deconstruct complex behaviors, and disrupt diseases that arise from secreted signaling proteins.