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. Microscopy and biochemical fractionation coupled with mass spectrometry 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 cells.

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.

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).

Gene Information eXtension (GIX): effortless retrieval of gene product information on any website.

Retrieving information about genes is a continuous and time-consuming aspect of systems biology. While there are resources that can fulfill this need, they require navigation to different and often complex websites, execution of a search (or searches) and selection of a result before the user can view pertinent information. To streamline this process, we developed a browser extension called GIX (Gene Information eXtension; https://gene-info.

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.

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).

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.

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.

Data Independent Acquisition analysis in ProHits 4.0.

Unlabelled: 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.

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.

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.

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.

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

Unlabelled: 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.

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.

Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kinase-phosphatase interactions.

The Hippo pathway regulates organ size and tissue homeostasis in response to multiple stimuli, including cell density and mechanotransduction. Pharmacological inhibition of phosphatases can also stimulate Hippo signaling in cell culture. We defined the Hippo protein-protein interaction network with and without inhibition of serine and threonine phosphatases by okadaic acid.

Profiling the kinome: current capabilities and future challenges.

Protein kinases are the second largest human protein family, but in terms of research interest, both basic and applied, they are surely the most popular. Over the past decade, many techniques and approaches for studying the kinome have been described and the pace of development is ever increasing. Presently, a molecular biologist can approach the kinome from many different angles: what kinases are active during a specific cell state of interest or become activated in response to a specific stimulus? What are the effects of controlling the activation status of an individual kinase? What substrates are targeted by a particular kinase, either in general or under particular conditions? And what kinase is responsible for targeting a specific phosphorylation site of interest? These are some of the more commonly asked questions during any kinase-centric research project and different strategies have been devised for answering such queries.

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.

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-3β (GSK-3β).

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.

Conservation, variability and the modeling of active protein kinases.

The human proteome is rich with protein kinases, and this richness has made the kinase of crucial importance in initiating and maintaining cell behavior. Elucidating cell signaling networks and manipulating their components to understand and alter behavior require well designed inhibitors. These inhibitors are needed in culture to cause and study network perturbations, and the same compounds can be used as drugs to treat disease.