Characterizing motifs of unknown function from the human proteome – OrphanMotifs

It has been estimated that 80% of all interactions formed between molecules of our cells are mediated by so-called short linear interaction motifs. Motifs are found within disordered regions of proteins and they mediate weak and transient interactions with their recognition domains. These properties make them almost completely invisible for conventional interactomic approaches. As a result, although we are able to predict functional motifs within protein sequences, they remain orphan with no identified interaction partners. Even though these putative motifs are uncharacterized, their biological importance is clearly implied, for example by their extreme sequence conservation, recurring disease-associated mutations, or viral hijacking. In this project, we will decipher the interactomes of thousands of the most important orphan motifs building on an innovative strategy developed by the principal investigator, called the native holdup (nHU). This revolutionary affinity-interactomic approach, in contrast to currently used interactomics methods, is fully capable to detect transient, motif-mediated interactions from cell extracts in a quantitative manner, by determining intrinsic affinities. We will use this method to identify cognate partners of orphan motifs, validate that these interactions can also form between full-length proteins, and we will determine under what conditions they form complexes in cells. Finally, we will measure how disease-related mutations targeting these motif-mediated interactions affect cellular phenotypes. This project will shed light on the partners of thousands of orphan motifs uncovered using computational approaches in the past decades. We are not only going to explore the interactions of these functional sites using a detailed quantitative-biophysical approach, but we will also show how these sites participate in key biological mechanisms revealing their importance in the pathophysiology of a multitude of disorders.