Panorama of ancient metazoan macromolecular complexes.

Macromolecular complexes are essential to conserved biological processes, but their prevalence across animals is unclear. By combining extensive biochemical fractionation with quantitative mass spectrometry, here we directly examined the composition of soluble multiprotein complexes among diverse metazoan models. Using an integrative approach, we generated a draft conservation map consisting of more than one million putative high-confidence co-complex interactions for species with fully sequenced genomes that encompasses functional modules present broadly across all extant animals. Clustering reveals a spectrum of conservation, ranging from ancient eukaryotic assemblies that have probably served cellular housekeeping roles for at least one billion years, ancestral complexes that have accrued contemporary components, and rarer metazoan innovations linked to multicellularity. We validated these projections by independent co-fractionation experiments in evolutionarily distant species, affinity purification and functional analyses. The comprehensiveness, centrality and modularity of these reconstructed interactomes reflect their fundamental mechanistic importance and adaptive value to animal cell systems.

Neuropilin-1 exerts co-receptor function for TGF-beta-1 on the membrane of cancer cells and enhances responses to both latent and active TGF-beta.

Neuropilin (Nrp)-1 and Nrp-2 are multifunctional proteins frequently expressed by cancer cells and contribute to tumor progression by mechanisms that are not well understood. They are co-receptors for vascular endothelial growth factor and class 3 semaphorins, but recently we found that Nrp1 also binds latent and active transforming growth factor (TGF)-ß1, and activates the latent form latency-associated peptide (LAP)-TGF-ß1. Here, we report that Nrp1 has affinity for TGF-ß receptors TßRI and TßRII, the signaling TGF-ß receptors, as well as TßRIII (betaglycan), as determined in binding assays, pull down assays and confocal microscopy. Nrp1 had a higher affinity for TßRI than TßRII and could form a complex with these receptors. In breast cancer cells, Nrp1 and TßRI cointernalized in the presence of TGF-ß1. Nrp1 acted as a TGF-ß co-receptor by augmenting canonical Smad2/3 signaling. Importantly, Nrp-positive cancer cells, unlike negative cells, were able to activate latent TGF-ß1 and respond. We examined two other membrane proteins that bind LAP-TGF-ß, i.e. an RGD-binding integrin (αvß3) and Glycoprotein A repetitions predominant (CLRRC32). RGD-binding integrins are frequently expressed by cancer cells, and glycoprotein A repetitions predominant is expressed by activated regulatory T cells that appear linked to poor tumor immunity. In vitro, these receptors did not activate LAP-TGF-ß1, but subsequent addition of Nrp1 activated the cytokine. Thus, Nrp1 might collaborate with other latent TGF-ß receptors in TGF-ß capture and activation. We also show that Nrp2 has activities similar to Nrp1. We conclude that Nrp1 is a co-receptor for TGF-ß1 and augments responses to latent and active TGF-ß. Since TGF-ß promotes metastasis this is highly relevant to cancer biology.

Proteome-wide dataset supporting the study of ancient metazoan macromolecular complexes.

Our analysis examines the conservation of multiprotein complexes among metazoa through use of high resolution biochemical fractionation and precision mass spectrometry applied to soluble cell extracts from 5 representative model organisms Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, Strongylocentrotus purpuratus, and Homo sapiens. The interaction network obtained from the data was validated globally in 4 distant species (Xenopus laevis, Nematostella vectensis, Dictyostelium discoideum, Saccharomyces cerevisiae) and locally by targeted affinity-purification experiments. Here we provide details of our massive set of supporting biochemical fractionation data available via ProteomeXchange (PXD002319-PXD002328), PPIs via BioGRID (185267); and interaction network projections via (http://metazoa.med.utoronto.ca) made fully accessible to allow further exploration. The datasets here are related to the research article on metazoan macromolecular complexes in Nature [1].

ComplexQuant: high-throughput computational pipeline for the global quantitative analysis of endogenous soluble protein complexes using high resolution protein HPLC and precision label-free LC/MS/MS.

The experimental isolation and characterization of stable multi-protein complexes are essential to understanding the molecular systems biology of a cell. To this end, we have developed a high-throughput proteomic platform for the systematic identification of native protein complexes based on extensive fractionation of soluble protein extracts by multi-bed ion exchange high performance liquid chromatography (IEX-HPLC) combined with exhaustive label-free LC/MS/MS shotgun profiling. To support these studies, we have built a companion data analysis software pipeline, termed ComplexQuant. Proteins present in the hundreds of fractions typically collected per experiment are first identified by exhaustively interrogating MS/MS spectra using multiple database search engines within an integrative probabilistic framework, while accounting for possible post-translation modifications. Protein abundance is then measured across the fractions based on normalized total spectral counts and precursor ion intensities using a dedicated tool, PepQuant. This analysis allows co-complex membership to be inferred based on the similarity of extracted protein co-elution profiles. Each computational step has been optimized for processing large-scale biochemical fractionation datasets, and the reliability of the integrated pipeline has been benchmarked extensively. This article is part of a Special Issue entitled: From protein structures to clinical applications.