Grant: Discovery Grants Program - Individual

Proteins are dominant drivers of biological processes underlying cellular physiology. They allow cells to interpret and respond to internal and external cues. Protein-protein interactions (PPIs) give rise to densely connected networks that govern all cellular processes. Large-scale studies to define the human protein interactome have catalogued many PPIs. However, hundreds of thousands of PPIs remain undiscovered. While most characterized PPIs reflect stable, high affinity interactions, the importance of dynamic, low-affinity PPIs mediated through intrinsically disordered protein regions is emerging. Most commonly, these are linear stretches of amino acids, often less than 10 residues, known as short linear motifs (SLiMs). SLiMs imake highly specific, yet transient interactions that impact virtually every aspect of cellular function. Current research demonstrates that no single approach is particularly well suited for identifying SLiM-based interactions, emphasizing the need for additional complementary experimental approaches to fill this gap in knowledge. Progress We have developed a novel "many-vs-many" genetic approach to identify SLiMs. We exploit protein nuclear import to screen a large number of peptides against endogenous nuclear proteins within a cellular context. While we designed this technology to function within the yeast S. cerevisiae, PPI domains, especially those involved in protein nuclear import, are highly conserved between yeast and mammals. Our system exploits endogenous cellular protein nuclear import as a means to select for short peptides that indirectly mediate nuclear import by interacting with a second endogenous protein containing an actual nuclear localization signal. Via this "piggybacking" transport mechanism, we can readily identify novel SLiMs that bind endogenous nuclear targeted proteins to gain access to the nucleus and activate reporter gene expression. At least some of these function in mammalian cells and can be identified in the human proteome. Objectives 1 Perform a large scale, high-throughput screen using a random peptide library to identify SLiMs mediating nuclear import 2 Identify peptides with similarity to mammalian proteins and evaluate their ability to mediate nuclear localization in a mammalian system 3 Identify PPIs mediated by the mammalian protein SLiM equivalent using mass spectrometry to identify their targets 4 Characterize motifs within their endogenous context Significance The "many-vs-many" approach we developed is economical and unique. It allows high throughput testing of random peptides against the yeast nuclear proteome in vivo. Identifying novel SLiMs and the PPIs they facilitate is fundamental to understanding many aspects of cellular biology. The new knowledge generated by these studies represents a potential resource in studies of fundamental biology and may have applications in synthetic biology.