A proximity-dependent biotinylation map of a human cell.

Compartmentalization is a defining characteristic of eukaryotic cells, and partitions distinct biochemical processes into discrete subcellular locations. Microscopy1 and biochemical fractionation coupled with mass spectrometry2-4 have defined the proteomes of a variety of different organelles, but many intracellular compartments have remained refractory to such approaches. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach to define the composition of cellular compartments in living cells5-7. Here we present a BioID-based map of a human cell on the basis of 192 subcellular markers, and define the intracellular locations of 4,145 unique proteins in HEK293 cells. Our localization predictions exceed the specificity of previous approaches, and enabled the discovery of proteins at the interface between the mitochondrial outer membrane and the endoplasmic reticulum that are crucial for mitochondrial homeostasis. On the basis of this dataset, we created humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to understand BioID results better.

Properties of Stress Granule and P-Body Proteomes.

Stress granules and P-bodies are cytosolic biomolecular condensates that dynamically form by the phase separation of RNAs and proteins. They participate in translational control and buffer the proteome. Upon stress, global translation halts and mRNAs bound to the translational machinery and other proteins coalesce to form stress granules (SGs). Similarly, translationally stalled mRNAs devoid of translation initiation factors shuttle to P-bodies (PBs). Here, we review the cumulative progress made in defining the protein components that associate with mammalian SGs and PBs. We discuss the composition of SG and PB proteomes, supported by a new user-friendly database (http://rnagranuledb.lunenfeld.ca/) that curates current literature evidence for genes or proteins associated with SGs or PBs. As previously observed, the SG and PB proteomes are biased toward intrinsically disordered regions and have a high propensity to contain primary sequence features favoring phase separation. We also provide an outlook on how the various components of SGs and PBs may cooperate to organize and form membraneless organelles.

Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains.

Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.

High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies.

mRNA processing, transport, translation, and ultimately degradation involve a series of dedicated protein complexes that often assemble into large membraneless structures such as stress granules (SGs) and processing bodies (PBs). Here, systematic in vivo proximity-dependent biotinylation (BioID) analysis of 119 human proteins associated with different aspects of mRNA biology uncovers 7424 unique proximity interactions with 1,792 proteins. Classical bait-prey analysis reveals connections of hundreds of proteins to distinct mRNA-associated processes or complexes, including the splicing and transcriptional elongation machineries (protein phosphatase 4) and the CCR4-NOT deadenylase complex (CEP85, RNF219, and KIAA0355). Analysis of correlated patterns between endogenous preys uncovers the spatial organization of RNA regulatory structures and enables the definition of 144 core components of SGs and PBs. We report preexisting contacts between most core SG proteins under normal growth conditions and demonstrate that several core SG proteins (UBAP2L, CSDE1, and PRRC2C) are critical for the formation of microscopically visible SGs.

MOB1 Mediated Phospho-recognition in the Core Mammalian Hippo Pathway.

The Hippo tumor suppressor pathway regulates organ size and tissue homoeostasis in response to diverse signaling inputs. The core of the pathway consists of a short kinase cascade: MST1 and MST2 phosphorylate and activate LATS1 and LATS2, which in turn phosphorylate and inactivate key transcriptional coactivators, YAP1 and TAZ (gene WWTR1). The MOB1 adapter protein regulates both phosphorylation reactions firstly by concurrently binding to the upstream MST and downstream LATS kinases to enable the trans phosphorylation reaction, and secondly by allosterically activating the catalytic function of LATS1 and LATS2 to directly stimulate phosphorylation of YAP and TAZ. Studies of yeast Mob1 and human MOB1 revealed that the ability to recognize phosphopeptide sequences in their interactors, Nud1 and MST2 respectively, was critical to their roles in regulating the Mitotic Exit Network in yeast and the Hippo pathway in metazoans. However, the underlying rules of phosphopeptide recognition by human MOB1, the implications of binding specificity for Hippo pathway signaling, and the generality of phosphopeptide binding function to other human MOB family members remained elusive.Employing proteomics, peptide arrays and biochemical analyses, we systematically examine the phosphopeptide binding specificity of MOB1 and find it to be highly complementary to the substrate phosphorylation specificity of MST1 and MST2. We demonstrate that autophosphorylation of MST1 and MST2 on several threonine residues provides multiple MOB1 binding sites with varying binding affinities which in turn contribute to a redundancy of MST1-MOB1 protein interactions in cells. The crystal structures of MOB1A in complex with two favored phosphopeptide sites in MST1 allow for a full description of the MOB1A phosphopeptide-binding consensus. Lastly, we show that the phosphopeptide binding properties of MOB1A are conserved in all but one of the seven MOB family members in humans, thus providing a starting point for uncovering their elusive cellular functions.

SAINTq: Scoring protein-protein interactions in affinity purification - mass spectrometry experiments with fragment or peptide intensity data.

SAINT (Significance Analysis of INTeractome) is a probabilistic method for scoring bait-prey interactions against negative controls in affinity purification - mass spectrometry (AP-MS) experiments. Our published SAINT algorithms use spectral counts or protein intensities as the input for calculating the probability of true interaction, which enables objective selection of high-confidence interactions with false discovery control. With the advent of new protein quantification methods such as Data Independent Acquisition (DIA), we redeveloped the scoring method to utilize the reproducibility information embedded in the peptide or fragment intensity data as a key scoring criterion, bypassing protein intensity summarization required in the previous SAINT workflow. The new software package, SAINTq, addresses key issues in the interaction scoring based on intensity data, including treatment of missing values and selection of peptides and fragments for scoring each prey protein. We applied SAINTq to two independent DIA AP-MS data sets profiling the interactome of MEPCE and EIF4A2 and that of 14-3-3ß, and benchmarked the performance in terms of recovering previously reported literature interactions in the iRefIndex database. In both data sets, the SAINTq analysis using the fragment-level intensity data led to the most sensitive detection of literature interactions at the same level of specificity. This analysis outperforms the analysis using protein intensity data summed from fragment intensity data that is equivalent to the model in SAINTexpress.

An unbiased proteomic screen reveals caspase cleavage is positively and negatively regulated by substrate phosphorylation.

Post-translational modifications of proteins regulate diverse cellular functions, with mounting evidence suggesting that hierarchical cross-talk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death via cleavage of key structural and enzymatic proteins that in some instances is inhibited by phosphorylation near the scissile bond. In this study, we systematically investigated how protein phosphorylation affects susceptibility to caspase cleavage using an N-terminomic strategy, namely, a modified terminal amino isotopic labeling of substrates (TAILS) workflow, to identify proteins for which caspase-catalyzed cleavage is modulated by phosphatase treatment. We validated the effects of phosphorylation on three of the identified proteins and found that Yap1 and Golgin-160 exhibit decreased cleavage when phosphorylated, whereas cleavage of MST3 was promoted by phosphorylation. Furthermore, using synthetic peptides we systematically examined the influence of phosphoserine throughout the entirety of caspase-3, -7, and -8 recognition motifs and observed a general inhibitory effect of phosphorylation even at residues considered outside the classical consensus motif. Overall, our work demonstrates a role for phosphorylation in controlling caspase-mediated cleavage and shows that N-terminomic strategies can be tailored to study cross-talk between phosphorylation and proteolysis.

A web-tool for visualizing quantitative protein-protein interaction data.

Quantitative interaction proteomics data can be a challenge to efficiently analyze and subsequently present to an audience in a simple and easy to understand format that still conveys sufficient levels of information. Here we present freely accessible and open-source web tools for displaying multiple parameters from quantitative protein-protein interaction data sets in a visually intuitive format. Given a set of "bait" proteins with detected "prey" interactions, dot plots can be generated to display absolute spectral counts for the preys, relative spectral counts between baits and confidence levels for the interactions (e.g. as determined by SAINTexpress). Additional tools are available for displaying fold change results between numerous baits with their associated confidence level (e.g. resulting from intensity measurements) and pairwise bait analyses displaying spectral counts, confidence score and fold change differences in a scatter plot format. These tools make it easy for the user to identify important interaction changes, interpret their data, and present this information to others in an intuitive way.

STK25 protein mediates TrkA and CCM2 protein-dependent death in pediatric tumor cells of neural origin.

The TrkA receptor tyrosine kinase induces death in medulloblastoma cells via an interaction with the cerebral cavernous malformation 2 (CCM2) protein. We used affinity proteomics to identify the germinal center kinase class III (GCKIII) kinases STK24 and STK25 as novel CCM2 interactors. Down-modulation of STK25, but not STK24, rescued medulloblastoma cells from NGF-induced TrkA-dependent cell death, suggesting that STK25 is part of the death-signaling pathway initiated by TrkA and CCM2. CCM2 can be phosphorylated by STK25, and the kinase activity of STK25 is required for death signaling. Finally, STK25 expression in tumors is correlated with positive prognosis in neuroblastoma patients. These findings delineate a death-signaling pathway downstream of neurotrophic receptor tyrosine kinases that may provide targets for therapeutic intervention in pediatric tumors of neural origin.

Biochemical, proteomic, structural, and thermodynamic characterizations of integrin-linked kinase (ILK): cross-validation of the pseudokinase.

Integrin-linked kinase (ILK) is one of the few evolutionarily conserved focal adhesion proteins involved in diverse cell adhesion-dependent physiological and pathological responses. Despite more than a decade of studies and extensive literature, the kinase function of ILK is controversial. ILK contains a highly degraded kinase active site but it has been argued that ILK may be an unusual manganese (Mn)-dependent serine-threonine kinase that targets specific substrates such as glycogen synthase kinase-3ß (GSK-3ß). In this study, we have tackled this issue by a systematic bottom-up biochemical, proteomic, structural, and thermodynamic analysis of ILK. We show that recombinant ILK from either bacteria or mammalian cells exhibits no kinase activity on GSK-3ß in the presence of either Mn(2+) or the conventional kinase co-factor Mg(2+). A comprehensive and unbiased whole cell-based kinase assay using entire mammalian CG-4 and C2C12 cell lysate did not identify any specific ILK substrates. High resolution crystallographic structure analysis further confirmed that the Mn-bound ILK adopts the same pseudo active site conformation as that of the Mg-bound ILK. More importantly, thermodynamic analysis revealed that the K220M mutation, previously thought to inactivate ILK by disrupting ATP binding, significantly impairs the structural integrity and stability of ILK, which provides a new basis for understanding how this mutation caused renal agenesis, a failure of fetal kidney development. Collectively, our data provide strong evidence that ILK lacks intrinsic kinase function. It is a bona fide pseudokinase that likely evolved from an ancestral catalytic counterpart to act as a distinct scaffold to mediate protein-protein interactions during focal adhesion assembly and many other cellular events.

The GATOR-Rag GTPase pathway inhibits mTORC1 activation by lysosome-derived amino acids.

The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR-Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase-independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.

The role of conserved water molecules in the catalytic domain of protein kinases.

Protein kinases are essential signaling molecules with a characteristic bilobal shape that has been studied for over 15 years. Despite the number of crystal structures available, little study has been directed away from the prototypical functional elements of the kinase domain. We have performed a structural alignment of 13 active-conformation kinases and discovered the presence of six water molecules that occur in conserved locations across this group of diverse kinases. Molecular dynamics simulations demonstrated that these waters confer a great deal of stability to their local environment and to a key catalytic residue. Our results highlight the importance of novel elements within the greater kinase family and suggest that conserved water molecules are necessary for efficient kinase function.

Data Independent Acquisition analysis in ProHits 4.0.

Affinity purification coupled with mass spectrometry (AP-MS) is a powerful technique for the identification and quantification of physical interactions. AP-MS requires careful experimental design, appropriate control selection and quantitative workflows to successfully identify bona fide interactors amongst a large background of contaminants. We previously introduced ProHits, a Laboratory Information Management System for interaction proteomics, which tracks all samples in a mass spectrometry facility, initiates database searches and provides visualization tools for spectral counting-based AP-MS approaches. More recently, we implemented Significance Analysis of INTeractome (SAINT) within ProHits to provide scoring of interactions based on spectral counts. Here, we provide an update to ProHits to support Data Independent Acquisition (DIA) with identification software (DIA-Umpire and MSPLIT-DIA), quantification tools (through DIA-Umpire, or externally via targeted extraction), and assessment of quantitative enrichment (through mapDIA) and scoring of interactions (through SAINT-intensity). With additional improvements, notably support of the iProphet pipeline, facilitated deposition into ProteomeXchange repositories and enhanced export and viewing functions, ProHits 4.0 offers a comprehensive suite of tools to facilitate affinity proteomics studies. SIGNIFICANCE: It remains challenging to score, annotate and analyze proteomics data in a transparent manner. ProHits was previously introduced as a LIMS to enable storing, tracking and analysis of standard AP-MS data. In this revised version, we expand ProHits to include integration with a number of identification and quantification tools based on Data-Independent Acquisition (DIA). ProHits 4.0 also facilitates data deposition into public repositories, and the transfer of data to new visualization tools.

Phenotypic and Interaction Profiling of the Human Phosphatases Identifies Diverse Mitotic Regulators.

Reversible phosphorylation is a fundamental regulatory mechanism, intricately coordinated by kinases and phosphatases, two classes of enzymes widely disrupted in human disease. To better understand the functions of the relatively understudied phosphatases, we have used complementary affinity purification and proximity-based interaction proteomics approaches to generate a physical interactome for 140 human proteins harboring phosphatase catalytic domains. We identified 1,335 high-confidence interactions (1,104 previously unreported), implicating these phosphatases in the regulation of a variety of cellular processes. Systematic phenotypic profiling of phosphatase catalytic and regulatory subunits revealed that phosphatases from every evolutionary family impinge on mitosis. Using clues from the interactome, we have uncovered unsuspected roles for DUSP19 in mitotic exit, CDC14A in regulating microtubule integrity, PTPRF in mitotic retraction fiber integrity, and DUSP23 in centriole duplication. The functional phosphatase interactome further provides a rich resource for ascribing functions for this important class of enzymes.

Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes.

Mapping protein-protein interactions for chromatin-associated proteins remains challenging. Here we explore the use of BioID, a proximity biotinylation approach in which a mutated biotin ligase (BirA*) is fused to a bait of interest, allowing for the local activation of biotin and subsequent biotinylation of proteins in the bait vicinity. BioID allowed for successful interactome mapping of core histones and members of the mediator complex. We explored the background signal produced by the BioID approach and found that using distinct types of controls increased the stringency of our statistical analysis with SAINTexpress. A direct comparison of BioID with our AP-MS protocol optimized for chromatin-associated protein complexes revealed that the approaches identified few shared interaction partners and enriched for distinct biological processes; yet, both approaches permitted the recovery of biologically meaningful interactions. While no clear bias could be observed for either technique toward protein complexes of particular functions, BioID allowed for the purification of proteins of lower cellular abundance. Finally, we were able to identify a strong association of MED4 with the centrosome by BioID and validated this finding by immunofluorescence. In summary, BioID complements AP-MS for the study of chromatin-associated protein complexes. BIOLOGICAL SIGNIFICANCE: This manuscript describes the application of BioID, a proximity biotinylation approach, to chromatin-associated proteins, namely core histones and members of the mediator complex. We observed that BioID was successful at identifying known interaction partners for the baits tested, but also allowed novel putative interaction partners to be identified. By performing a detailed comparison of BioID versus a standard method for interactome mapping (affinity purification coupled to mass spectrometry, AP-MS), we show that the approaches were complementary, allowing for purification of different interaction partners. These interaction partners were different in the biological processes they are associated with, but also in their abundance. BioID represents a significant technical development in the field of chromatin research by expanding the search space for interactome mapping beyond what is possible with AP-MS. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.

CCM-3/STRIPAK promotes seamless tube extension through endocytic recycling.

The mechanisms governing apical membrane assembly during biological tube development are poorly understood. Here, we show that extension of the C. elegans excretory canal requires cerebral cavernous malformation 3 (CCM-3), independent of the CCM1 orthologue KRI-1. Loss of ccm-3 causes canal truncations and aggregations of canaliculular vesicles, which form ectopic lumen (cysts). We show that CCM-3 localizes to the apical membrane, and in cooperation with GCK-1 and STRIPAK, promotes CDC-42 signalling, Golgi stability and endocytic recycling. We propose that endocytic recycling is mediated through the CDC-42-binding kinase MRCK-1, which interacts physically with CCM-3-STRIPAK. We further show canal membrane integrity to be dependent on the exocyst complex and the actin cytoskeleton. This work reveals novel in vivo roles of CCM-3·STRIPAK in regulating tube extension and membrane integrity through small GTPase signalling and vesicle dynamics, which may help explain the severity of CCM3 mutations in patients.