An Atlas of human kinase regulation

The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision-making has been limited by the small number of simultaneously monitored phospho- regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 condi- tions from a compilation of nearly 3 million phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co-regulation along the conditions predicts kinase-complex and kinase-substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essen- tial mediators of stem cell differentiation, modulators of Salmonella infection and new targets of AKT1. This provides a global view of human phosphorylation-based signaling and the necessary context to better understand kinase driven decision-making

Species-specific activity of antibacterial drug combinations

International audienceThe spread of antimicrobial resistance has become a serious public health concern, making once treatable diseases deadly again and undermining breakthrough achievements of modern medicine 1,2. Drug combinations can aid in fighting multi-drug resistant (MDR) bacterial infections, yet, are largely unexplored and rarely used in clinics. To identify general principles for antibacterial drug combinations and understand their potential, we profiled ~3,000 dose-resolved combinations of antibiotics, human-targeted drugs and food additives in 6 strains from three Gram-negative pathogens, Escherichia coli, Salmonella Typhimurium and Pseudomonas aeruginosa. Despite their phylogenetic relatedness, more than 70% of the detected drug-drug interactions are species-specific and 20% display strain specificity, revealing a large potential for narrow-spectrum therapies. Overall, antagonisms are more common than synergies and occur almost exclusively between drugs targeting different cellular processes, whereas synergies are more conserved and enriched in drugs targeting the same process. We elucidate mechanisms underlying this dichotomy and further use our resource to dissect the interactions of the food additive, vanillin. Finally, we demonstrate that several synergies are effective against MDR clinical isolates in vitro and during Galleria mellonella infections with one reverting resistance to the last-resort antibiotic, colistin

Characterization of a novel protein translocation system found ubiquitously in the membranes of Gram-negative bacteria

Bacteria have evolved complex protein machinery to secrete proteins into the environment. In Gram-negative bacteria, secreted substrates require efficient targeting and translocation across the inner- and outer-membrane by protein secretion systems. The Omp85 protein superfamily is evolutionarily conserved and functions by translocating substrates into, or across, the outer membrane of Gram-negative bacteria. This thesis describes the discovery of a novel protein secretion machine that is required for the efficient delivery of a group of surface exposed proteins called autotransporters. We have named this protein secretion system the translocation and assembly module (TAM) which is composed of the Omp85 outer membrane component TamA and the inner membrane protein TamB which together interact to form a complex spanning the periplasmic space. Chapter three describes the genomic relationship between tamA and tamB and a bioinformatic analysis of the protein subdomains. The protein products within the operon were then localized and their membrane topology determined. Chapter 4 presents the protein-protein interactions within the TAM complex. Finally, determination of the function of the TAM complex and the requirements for particular subdomains is presented in Chapter 5. Together, these findings have uncovered a novel protein secretion system within the membranes of Gram-negative bacteria. Further, this work has elucidated the function of this protein complex as a mediator of efficient secretion of autotransporters. Due to the virulence associate functions of autotransporter substrates, therapeutic inhibition of the TAM complex may prove to be a viable pursuit to inhibit Gram-negative bacterial infections

Assembly of β-barrel proteins into bacterial outer membranes

Membrane proteins with a β-barrel topology are found in the outer membranes of Gram-negative bacteria and in the plastids and mitochondria of eukaryotic cells. The assembly of these membrane proteins depends on a protein folding reaction (to create the

Evolution of the Translocation and Assembly Module (TAM)

<p>Supporting data to 'Evolution of the Translocation and Assembly Module (TAM)' Heinz E, Selkrig J, Belousoff M, Lithgow T. 2015 Genome Biology and Evolution</p> <p> </p

Sequence and Structure-Based Analysis of Specificity Determinants in Eukaryotic Protein Kinases

Protein kinases lie at the heart of cell-signaling processes and are often mutated in disease. Kinase target recognition at the active site is in part determined by a few amino acids around the phosphoacceptor residue. However, relatively little is known about how most preferences are encoded in the kinase sequence or how these preferences evolved. Here, we used alignment-based approaches to predict 30 specificity-determining residues (SDRs) for 16 preferences. These were studied with structural models and were validated by activity assays of mutant kinases. Cancer mutation data revealed that kinase SDRs are mutated more frequently than catalytic residues. We have observed that, throughout evolution, kinase specificity has been strongly conserved across orthologs but can diverge after gene duplication, as illustrated by the G protein-coupled receptor kinase family. The identified SDRs can be used to predict kinase specificity from sequence and aid in the interpretation of evolutionary or disease-related genomic variants.ISSN:2666-3864ISSN:2211-124

An atlas of human kinase regulation

The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision-making has been limited by the small number of simultaneously monitored phospho-regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 conditions derived from a large compilation of phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co-regulation along the conditions predicts kinase–complex and kinase–substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essential mediators of stem cell differentiation, modulators of Salmonella infection, and new targets of AKT1. This provides a global view of human phosphorylation-based signaling and the necessary context to better understand kinase-driven decision-making.ISSN:1744-429

Systematic analysis of drug combinations against Gram-positive bacteria

Drug combinations can expand options for antibacterial therapies but have not been systematically tested in Gram-positive species. We profiled ~8,000 combinations of 65 antibacterial drugs against the model species Bacillus subtilis and two prominent pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Thereby, we recapitulated previously known drug interactions, but also identified ten times more novel interactions in the pathogen S. aureus, including 150 synergies. We showed that two synergies were equally effective against multidrug-resistant S. aureus clinical isolates in vitro and in vivo. Interactions were largely species-specific and synergies were distinct from those of Gram-negative species, owing to cell surface and drug uptake differences. We also tested 2,728 combinations of 44 commonly prescribed non-antibiotic drugs with 62 drugs with antibacterial activity against S. aureus and identified numerous antagonisms that might compromise the efficacy of antimicrobial therapies. We identified even more synergies and showed that the anti-aggregant ticagrelor synergized with cationic antibiotics by modifying the surface charge of S. aureus. All data can be browsed in an interactive interface (https://apps.embl.de/combact/).</p