Our research focuses on the functional role of the intrinsically disordered regions (IDRs) of the proteome. Specifically, we utilise evolutionary, proteomic and genomic data to gain a better understanding of the function and regulation of SLiM-mediated interaction interfaces. We aim to answer two major open questions about intrinsically disordered regions: (i) what are the modules that are responsible for their functionality and (ii) how do perturbations in the cell modulate the functionality of these modules.
What are the modules that are responsible for their functionality of IDRs?
Approximately one-third of the human proteome consists of IDRs. However, the functional role of the vast majority of these regions is unknown. The most common functional modules within IDRs are the compact, linear protein interaction sites known as short linear motifs (SLiMs). The human proteome has been estimated to contain more than a hundred thousand – and possibly up to a million - SLiM instances. This would make them the most numerous protein modules in the cell.
Yet, to date, only few thousand instances are known. A key goal of our research is the development of an in silico framework for the discovery of novel SLiM instances. We develop three SLiM discovery tools, PSSMSearch, SLiMSearch and SLiMPrints. PSSMSearch and SLiMSearch screen whole proteomes for novel instances of known motifs using motif specificity determinants and incorporate ancillary evolutionary, proteomic and genomic data to rank putative motifs instances by confidence of functionality.
SLiMPrints discovers novel motifs by searching for groupings of residues that are under greater functional constraint than their surrounding residues, a strong functional discriminator for motifs. We also develop the proteomic phage display (Pro-PD) in vitro protein-protein interaction discovery method - the first high-throughput experimental method for short, linear motif discovery.
Pro-PD leverages advances in high-throughput oligonucleotide synthesis to create large designed libraries of peptides displayed in the surface coat proteins of bacteriophage and permits direct interrogation of biological peptides on a proteome-wide scale. The development of Pro-PD is part of a long-running collaboration with the Ivarsson Group at the Uppsala Universitet.
How do perturbations in the cell modulate the functionality of IDRs?
We analyse the intrinsic attributes of SLiMs that predispose them to both pre- and post-translational regulation to discover novel conditional mechanisms controlling SLiM function. The relatively weak affinity of SLiM-containing interfaces can be easily modulated by post-translational modification (PTM) and, consequently, modification of a residue in or adjacent to a linear motif is a common mechanism to conditionally and dynamically regulate SLiM-mediated interactions.
The compact footprint of SLiMs facilitates the occurrence of regions with high functional density containing multiple adjacent or overlapping motifs. These multi-module interfaces can act cooperatively or competitively in a highly controlled manner to conditionally build functionally distinct complexes depending on the local abundance of the binding partners.
Finally, the inclusion or exclusion of SLiM-containing exons by pre-translational mechanisms such as alternative splicing, alternative promoter usage and RNA editing can rewire the interaction network of a protein isoform. These three mechanisms can modulate SLiM function in a cell state- or tissue-specific manner, thereby altering its sub-cellular localization, half-life, binding partners, activity or modification state, and hence its function.
To date, a substantial number of conditional SLiM-mediated interactions have been biochemically characterized, highlighting the central role that pre and post-transcriptional modulation of SLiMs plays in the regulation of dynamic cellular processes.