Cell activity is the result of a series of biological processes. In this context, the protein-protein interaction (PPI) networks of signalling pathways play a vital role in mediating the entirety of cell activities due to their complexity and specificity (1). Pathogenic protein variants in key signaling proteins may lead to impairments of signalling pathways, which in turn will affect the orderly progress of cell activities. Meanwhile, non-pathogenic variants may also cause human individual differences, thereby causing phenotypic polymorphisms.

To assess the role of protein variants in modulating protein-protein interaction networks we focused on the PDZ domain family in signal proteins. The human proteome contains 266 PDZ domains from 155 proteins and more than 4,000 PDZ binding motifs (PBM). PDZ-PBM interaction networks regulate multiple biological processes such as transport, ion channel signalling, cell-cell junctions, cell polarity and adhesion, etc. Functional perturbations in these networks can cause the development of cancers, degenerations of the nervous system and other diseases (2-4).

We rationally designed 52 different variants from 5 wt PDZ domains, based both on allele frequency and structural criteria. All variants are part of the natural polymorphism, including disease-associated variants, human natural polymorphic variants and orthologs from close organisms. Different from common high-throughput qualitative assessments, we used a high-throughput quantitative approach developed in our group, Holdup assay with fluorescence readout, which quantifies PPI affinities (K_d) at an unprecedented scale and precision. We measured the affinities of the different PDZ for a set of 374 PBMs, and thus quantified a network of ~20,000 wt and variant PDZ-PBM interactions. We observed different types of interactome perturbations: unaffected interactome, destabilised interactome, isolated interactome changes, broad interactome reshuffling, etc. (Figure 1). Furthermore, these interactome perturbations depended on the variant categories: likely pathogenic variants vs. likely benign variants, variants in protein binding or non-binding sites, variants with different alleles frequencies.

The long-term plan of this project is to further understand the impact of single variants on cell activities through understanding the impact of single variants on PPI networks, and ultimately describe pathogenic mechanisms with better precision.

(1) Giancotti, "Complexity and specificity of integrin signalling", Nature Cell Biology, 2000, 2: E13–E14.

(2) Lee & Zeng, "PDZ domains and their binding partners: structure, specificity, and modification", Cell Communication and Signaling, 2010, 8: 8.

(3) Gogl, et al., "Quantitative fragmentomics allow affinity mapping of interactomes", Nature Communications, 2022, 13: 5472.

(4) Nardella, et al., "Targeting PDZ domains as potential treatment for viral infections, neurodegeneration and cancer", Biol Direct. 2021, 16: 15.