Proteins are major effectors and regulators of biological processes that can elicit multiple functions depending on their interaction with other proteins. The organization of proteins into ...macromolecular complexes and their quantitative distribution across these complexes is, therefore, of great biological and clinical significance. In this paper, we describe an integrated experimental and computational technique to quantify hundreds of protein complexes in a single operation. The method consists of size exclusion chromatography (SEC) to fractionate native protein complexes, SWATH/DIA mass spectrometry to precisely quantify the proteins in each SEC fraction, and the computational framework CCprofiler to detect and quantify protein complexes by error‐controlled, complex‐centric analysis using prior information from generic protein interaction maps. Our analysis of the HEK293 cell line proteome delineates 462 complexes composed of 2,127 protein subunits. The technique identifies novel sub‐complexes and assembly intermediates of central regulatory complexes while assessing the quantitative subunit distribution across them. We make the toolset CCprofiler freely accessible and provide a web platform, SECexplorer, for custom exploration of the HEK293 proteome modularity.
Synopsis
The study presents an integrated framework for targeted, complex‐centric analysis based on size exclusion chromatography (SEC) and SWATH/DIA mass spectrometry. The workflow facilitates the parallel detection of hundreds of protein complexes and their variants at high selectivity and under error‐control.
The presented workflow is based on the concept of complex‐centric proteome profiling and combines size exclusion chromatography (SEC) with SWATH/DIA mass spectrometry.
The implementation of the complex‐centric data analysis is supported by the computational framework CCprofiler.
Application of the SEC‐SWATH‐MS workflow to HEK293 cells led the detection and quantification of subunit distribution of 462 distinct protein complexes containing 2,127 proteins and identified novel complex variants such as assembly intermediates.
The interactive platform, SECexplorer is presented to support custom complex‐centric exploration of SEC‐SWATH‐MS datasets.
The study presents an integrated framework for targeted, complex‐centric analysis based on size exclusion chromatography (SEC) and SWATH/DIA mass spectrometry. The workflow facilitates the parallel detection of hundreds of protein complexes and their variants at high selectivity and under error‐control.
Cellular information processing via reversible protein phosphorylation requires tight control of the localization, activity, and substrate specificity of protein kinases, which to a large extent is ...accomplished by complex formation with other proteins. Despite their critical role in cellular regulation and pathogenesis, protein interaction information is available for only a subset of the 518 human protein kinases. Here we present a global proteomic analysis of complexes of the human CMGC kinase group. In addition to subgroup-specific functional enrichment and modularity, the identified 652 high-confidence kinase-protein interactions provide a specific biochemical context for many poorly studied CMGC kinases. Furthermore, the analysis revealed a kinase-kinase subnetwork and candidate substrates for CMGC kinases. Finally, the presented interaction proteome uncovered a large set of interactions with proteins genetically linked to a range of human diseases, including cancer, suggesting additional routes for analyzing the role of CMGC kinases in controlling human disease pathways.
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•A high-confidence interaction proteome for the human CMGC kinase family•Organization of human CMGC kinases in kinase-kinase subnetworks•Network inference revealed CMGC kinase-substrates modules•Discovery of 108 physical interactions between kinases and disease-associated proteins
Protein kinases exert their key role in cellular regulation as members of complexes, but current information on protein interactions is limited to a subset of the 518 human kinases. Gstaiger and colleagues now provide a high-confidence systematic analysis of complexes in the human CMGC kinase group. Besides uncovering interactions and regulatory modules, the study links CMGC kinases to a set of disease-associated proteins, suggesting a critical role for CMGC kinases in controlling human disease pathways.
Reproducibility in research can be compromised by both biological and technical variation, but most of the focus is on removing the latter. Here we investigate the effects of biological variation in ...HeLa cell lines using a systems-wide approach. We determine the degree of molecular and phenotypic variability across 14 stock HeLa samples from 13 international laboratories. We cultured cells in uniform conditions and profiled genome-wide copy numbers, mRNAs, proteins and protein turnover rates in each cell line. We discovered substantial heterogeneity between HeLa variants, especially between lines of the CCL2 and Kyoto varieties, and observed progressive divergence within a specific cell line over 50 successive passages. Genomic variability has a complex, nonlinear effect on transcriptome, proteome and protein turnover profiles, and proteotype patterns explain the varying phenotypic response of different cell lines to Salmonella infection. These findings have implications for the interpretation and reproducibility of research results obtained from human cultured cells.
Rapidly increasing availability of genomic data and ensuing identification of disease associated mutations allows for an unbiased insight into genetic drivers of disease development. However, ...determination of molecular mechanisms by which individual genomic changes affect biochemical processes remains a major challenge. Here, we develop a multilayered proteomic workflow to explore how genetic lesions modulate the proteome and are translated into molecular phenotypes. Using this workflow we determine how expression of a panel of disease-associated mutations in the Dyrk2 protein kinase alter the composition, topology and activity of this kinase complex as well as the phosphoproteomic state of the cell. The data show that altered protein-protein interactions caused by the mutations are associated with topological changes and affected phosphorylation of known cancer driver proteins, thus linking Dyrk2 mutations with cancer-related biochemical processes. Overall, we discover multiple mutation-specific functionally relevant changes, thus highlighting the extensive plasticity of molecular responses to genetic lesions.
Most catalytic, structural and regulatory functions of the cell are carried out by functional modules, typically complexes containing or consisting of proteins. The composition and abundance of these ...complexes and the quantitative distribution of specific proteins across different modules are therefore of major significance in basic and translational biology. However, detection and quantification of protein complexes on a proteome-wide scale is technically challenging. We have recently extended the targeted proteomics rationale to the level of native protein complex analysis (complex-centric proteome profiling). The complex-centric workflow described herein consists of size exclusion chromatography (SEC) to fractionate native protein complexes, data-independent acquisition mass spectrometry to precisely quantify the proteins in each SEC fraction based on a set of proteotypic peptides and targeted, complex-centric analysis where prior information from generic protein interaction maps is used to detect and quantify protein complexes with high selectivity and statistical error control via the computational framework CCprofiler (https://github.com/CCprofiler/CCprofiler). Complex-centric proteome profiling captures most proteins in complex-assembled state and reveals their organization into hundreds of complexes and complex variants observable in a given cellular state. The protocol is applicable to cultured cells and can potentially also be adapted to primary tissue and does not require any genetic engineering of the respective sample sources. At present, it requires ~8 d of wet-laboratory work, 15 d of mass spectrometry measurement time and 7 d of computational analysis.
Tissue homeostasis is controlled by signaling systems that coordinate cell proliferation, cell growth and cell shape upon changes in the cellular environment. Deregulation of these processes is ...associated with human cancer and can occur at multiple levels of the underlying signaling systems. To gain an integrated view on signaling modules controlling tissue growth, we analyzed the interaction proteome of the human Hippo pathway, an established growth regulatory signaling system. The resulting high‐resolution network model of 480 protein‐protein interactions among 270 network components suggests participation of Hippo pathway components in three distinct modules that all converge on the transcriptional co‐activator YAP1. One of the modules corresponds to the canonical Hippo kinase cassette whereas the other two both contain Hippo components in complexes with cell polarity proteins. Quantitative proteomic data suggests that complex formation with cell polarity proteins is dynamic and depends on the integrity of cell‐cell contacts. Collectively, our systematic analysis greatly enhances our insights into the biochemical landscape underlying human Hippo signaling and emphasizes multifaceted roles of cell polarity complexes in Hippo‐mediated tissue growth control.
Synopsis
Systematic characterization of the human Hippo pathway protein interactome by quantitative mass spectrometry generates a high‐resolution network of 480 interactions among 270 proteins and reveals three major modules linked to the transcriptional coactivator YAP1.
The interactome of human Hippo signaling provides a rich resource of high confidence protein interactions.
Network topology revealed three major modules containing phosphatases, kinases and cell polarity proteins and converging at the transcriptional coactivator YAP1.
A subset of protein phosphatase 1 complexes binds and activates YAP1.
Cell‐cell contacts control YAP1 transcriptional activity as well as YAP1 complex formation with proteins linked to cell polarity.
Systematic characterization of the human Hippo pathway protein interactome by quantitative mass spectrometry generates a high‐resolution network of 480 interactions among 270 proteins and reveals three major modules linked to the transcriptional coactivator YAP1.
Serine/threonine phosphatases such as PP1 lack substrate specificity and associate with a large array of targeting subunits to achieve the requisite selectivity. The tumour suppressor ASPP ...(apoptosis-stimulating protein of p53) proteins associate with PP1 catalytic subunits and are implicated in multiple functions from transcriptional regulation to cell junction remodelling. Here we show that Drosophila ASPP is part of a multiprotein PP1 complex and that PP1 association is necessary for several in vivo functions of Drosophila ASPP. We solve the crystal structure of the human ASPP2/PP1 complex and show that ASPP2 recruits PP1 using both its canonical RVxF motif, which binds the PP1 catalytic domain, and its SH3 domain, which engages the PP1 C-terminal tail. The ASPP2 SH3 domain can discriminate between PP1 isoforms using an acidic specificity pocket in the n-Src domain, providing an exquisite mechanism where multiple motifs are used combinatorially to tune binding affinity to PP1.
Protein kinases are essential for signal transduction and control of most cellular processes, including metabolism, membrane transport, motility, and cell cycle. Despite the critical role of kinases ...in cells and their strong association with diseases, good coverage of their interactions is available for only a fraction of the 535 human kinases. Here, we present a comprehensive mass-spectrometry-based analysis of a human kinase interaction network covering more than 300 kinases. The interaction dataset is a high-quality resource with more than 5,000 previously unreported interactions. We extensively characterized the obtained network and were able to identify previously described, as well as predict new, kinase functional associations, including those of the less well-studied kinases PIM3 and protein O-mannose kinase (POMK). Importantly, the presented interaction map is a valuable resource for assisting biomedical studies. We uncover dozens of kinase-disease associations spanning from genetic disorders to complex diseases, including cancer.
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•AP-MS confirms over 1,300 known and reveals over 5,000 kinase protein interactions•Functional contexts and regulatory circuits for poorly studied kinases•Kinases are biochemically linked to a broad range of Mendelian disease modules•Biochemical context for kinase complexes containing human cancer proteins
In this issue of Molecular Cell, Buljan et al. present a comprehensive account of human kinase complexes for inferring new kinase functions, kinase signaling modules, and kinase complexes linked to genetic diseases.
The characterization of all protein complexes of human cells under defined physiological conditions using affinity purification-mass spectrometry (AP-MS) is a highly desirable step in the quest to ...understand the phenotypic effects of genomic information. However, such a challenging goal has not yet been achieved, as it requires reproducibility of the experimental workflow and high data consistency across different studies and laboratories. We systematically investigated the reproducibility of a standardized AP-MS workflow by performing a rigorous interlaboratory comparative analysis of the interactomes of 32 human kinases. We show that it is possible to achieve high interlaboratory reproducibility of this standardized workflow despite differences in mass spectrometry configurations and subtle sample preparation-related variations and that combination of independent data sets improves the approach sensitivity, resulting in even more-detailed networks. Our analysis demonstrates the feasibility of obtaining a high-quality map of the human protein interactome with a multilaboratory project.
Abstract
Myelofibrosis is a hematopoietic stem cell disorder belonging to the myeloproliferative neoplasms. Myelofibrosis patients frequently carry driver mutations in either
JAK2
or Calreticulin (
...CALR
) and have limited therapeutic options. Here, we integrate ex vivo drug response and proteotype analyses across myelofibrosis patient cohorts to discover targetable vulnerabilities and associated therapeutic strategies. Drug sensitivities of mutated and progenitor cells were measured in patient blood using high-content imaging and single-cell deep learning-based analyses. Integration with matched molecular profiling revealed three targetable vulnerabilities. First,
CALR
mutations drive BET and HDAC inhibitor sensitivity, particularly in the absence of high Ras pathway protein levels. Second, an MCM complex-high proliferative signature corresponds to advanced disease and sensitivity to drugs targeting pro-survival signaling and DNA replication. Third, homozygous
CALR
mutations result in high endoplasmic reticulum (ER) stress, responding to ER stressors and unfolded protein response inhibition. Overall, our integrated analyses provide a molecularly motivated roadmap for individualized myelofibrosis patient treatment.
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