23rd Human Proteome Organization World Congress

Functional proteomics and induced proximity

Keynote speaker

Keynote lecture

The surfaceome architecture of a human B cell

Organisation of the Proteome (PPI)

Cellular function is orchestrated by extracellular signaling cues to elicit intracellular responses to stimuli. Cell surface residing proteins, collectively termed the surfaceome, fulfill their function as signal transducers within protein communities. This cell surface receptor interaction network is complex and dynamic, forming a highly organized surfaceome architecture essential for cellular functionality. Exploring the lateral proximity network of cell surface residing proteins facilitates the understanding of the surfaceome architecture, hence allowing for manipulation by utilizing bivalent therapeutics, such as bispecific antibodies. Targeting cells based on specific combinations of proteins present on their surface holds great potential in the search for selecting disease-driving sub-populations. We set out to identify novel target combinations for such therapies by elucidating the surfaceome architecture of B cells. To probe the spatial surfaceome, we utilized the in-house developed technology LUX-MS, enabling light-mediated proximity detection of dynamic protein interactions on the cell surface. Small molecule Singlet Oxygen Generators (SOGs) facilitate light-mediated photo-oxidation of cell surface proteins in the nanometer vicinity. When delivered through a ligand-of-interest, SOGs enable the capture of receptor proximity networks. LUX-MS of the mature B cell marker IgM revealed proximity candidates, of which 15 were initially chosen for individual LUX-MS experiments to confirm their proximity to IgM in a reciprocal approach. Some of these candidates exhibit similar proximity networks to IgM, such as the co-receptor CD79a, but others display very distinct organizations, as in the case of the costimulatory molecule and CD27 ligand CD70. The extensive proximity network of CD70 suggests a bystander functionality due to its high expression levels but can also hint at distinct organizational hubs on a single cell level. Intersecting and distinct proximity networks of individual target receptors facilitate the establishment of a global view of the B cell surfaceome architecture through reciprocal network walking. Having established initial architectural insight into the B cell surfaceome landscape, we are currently expanding our datasets with a large set of CD-annotated receptor targets in an effort to generate a comprehensive view of the B cell surfaceome architecture. Establishing a model for the B cell surfaceome architecture offers an opportunity to expand the toolbox for improved targeted immunotherapy by revealing drug candidate pairs.

Oral presentation

Mapping of the human solute carrier transporter protein interaction landscape

Solute carrier (SLC) transporters form a superfamily of membrane proteins facilitating transmembrane transport of a wide range of substrates. SLCs play a vital role in cellular homeostasis and metabolic regulation, as they control nutrient, ion and drug transport. They consist of about 450 members which locate in cellular membranes including subcellular compartments. To achieve fine-tuned transport functions necessary for proper metabolic homeostasis, transporters act in concert with numerous other membrane, cytoskeletal and proteostatic proteins.More than half lack a well described interactome. We applied an AP-MS protocol tailored to transmembrane proteins to map the protein interaction network for more than 400 solute carriers. We further devised a ML-based scoring approach integrating scores of commonly used scoring tools (e.g. SAINT, CompPASS) and multiple quantitative and uniqueness features. The generated interactome covers more than 18"000 novel high-confidence protein interactions, therefore providing a basis for understanding the molecular regulation of this protein family.Clustering and functional enrichment analysis revealed interaction partners throughout the life span of the transporters. The extracted subnetworks allowed us to link interactors to proteostatic regulation pathways, including protein translation and posttranslational modification processes. In addition, we identified close to stoichiometric interactions, revealing known and novel stable assemblies of SLCs with chaperone-proteins.We performed a large-scale validation approach focusing on protein stability and localization changes upon genetic perturbation of prey proteins by RNAi. For 135 bait-prey combinations either approach was tested, with 40% showing a functional effect on the target SLC.Our dataset empowers the identification of core regulatory modules in which SLCs are involved and further contributes to the understanding of the biological roles of this understudied family of proteins.

Dynamic proximal interaction networks of stress granules during assembly and disassembly reveal RNA regulation important for stress granule formation.

Stress granules (SGs) are biomolecular condensates that transiently assemble during cellular stress and assist in the cellular stress response. SG formation is thought to be driven by multivalent interactions between proteins and transcripts released from translation machineries. Efforts to elucidate the mechanisms underlying SG formation has revealed the importance of protein-protein, protein-RNA and RNA-RNA interactions, which involve protein and RNA scaffolds. However, we lack in understanding additional mechanisms required for SG formation. Here, we present quantitative analyses of multi-bait SG proximity labeling (i.e., BioID) experiments performed during stress time course paralleled with a genome-wide chemical genetic interaction screen. First, we implemented pulse biotin labeling of SG proteome during stress and recovery, augmented by data-independent acquisition (DIA) method. Experimentally, we used a group of bait proteins that effectively label over 50% of the known SG proteome and performed BioID labeling during two different stress conditions (oxidative and hyperosmotic) that induce SG via distinct mechanisms. Through SAINT analysis, we identify ~1,300 high-confident proximal interactors (preys) in steady state, stress, and recovery periods, and predict that about a third of these preys represent SG proteome in either condition. To measure quantitative changes in the SG protein interaction network during SG assembly and disassembly, we utilized DIA-NN analyses to obtain protein abundance and exploited ANOVA tests to determine statistically significant changes. About 20% of preys show significant quantitative changes during assembly and disassembly of SGs, representing a pool of dynamic associations and dissociations, while ~80% remain the same. Regardless of the type of stressor, we observe dynamic proximal interaction changes between SG baits and the components of the CCR4-NOT deadenylase, global regulator of polyA tail lengths. In parallel to the proteomics screening, we utilized a small molecule that can inhibit SG formation, called lipoamide, to interrogate mechanisms underlying SG formation. We identified that CCR4-NOT structural components, CNOT1, CNOT2 and CNOT3, are required for lipoamide inhibitory action on SGs. Based on the function of CCR4-NOT complex, we propose that lengthening of poly(A) tails by loss of CCR4-NOT complex lowers the threshold for phase separation through increasing valency and overcomes the inhibitory effect of lipoamide. Together, we provide dynamic map of SG proximal interaction networks during SG formation and dissolution and propose that regulation of polyA lengths of mRNAs by CCR4-NOT complex is important for SG formation.

Plenary hall

Session

Organisation of the proteome

Session
Organisation of the proteome

Appointment

Chairs

Program

SessionOrganisation of the proteome

Appointment

Chairs

Program

Session
Organisation of the proteome