Reversible lysine-targeted probes reveal residence time-based kinase selectivity.

The expansion of the target landscape of covalent inhibitors requires the engagement of nucleophiles beyond cysteine. Although the conserved catalytic lysine in protein kinases is an attractive candidate for a covalent approach, selectivity remains an obvious challenge. Moreover, few covalent inhibitors have been shown to engage the kinase catalytic lysine in animals.

Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells.

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein-coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase-dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined.

A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition.

During early G1 phase, Rb is exclusively mono-phosphorylated by cyclin D:Cdk4/6, generating 14 different isoforms with specific binding patterns to E2Fs and other cellular protein targets. While mono-phosphorylated Rb is dispensable for early G1 phase progression, interfering with cyclin D:Cdk4/6 kinase activity prevents G1 phase progression, questioning the role of cyclin D:Cdk4/6 in Rb inactivation. To dissect the molecular functions of cyclin D:Cdk4/6 during cell cycle entry, we generated a single cell reporter for Cdk2 activation, RB inactivation and cell cycle entry by CRISPR/Cas9 tagging endogenous p27 with mCherry.

Phosphoproteomic analysis of protease-activated receptor-1 biased signaling reveals unique modulators of endothelial barrier function.

Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling.

Quantitative Multiplex Substrate Profiling of Peptidases by Mass Spectrometry.

Proteolysis is an integral component of life and has been implicated in many disease processes. To improve our understanding of peptidase function, it is imperative to develop tools to uncover substrate specificity and cleavage efficiency. Here, we combine the quantitative power of tandem mass tags (TMTs) with an established peptide cleavage assay to yield quantitative Multiplex Substrate Profiling by Mass Spectrometry (qMSP-MS).

Essential metabolism for a minimal cell.

JCVI-syn3A, a robust minimal cell with a 543 kbp genome and 493 genes, provides a versatile platform to study the basics of life. Using the vast amount of experimental information available on its precursor, , we assembled a near-complete metabolic network with 98% of enzymatic reactions supported by annotation or experiment. The model agrees well with genome-scale in vivo transposon mutagenesis experiments, showing a Matthews correlation coefficient of 0.

Identification of Common and Rare Genetic Variation Associated With Plasma Protein Levels Using Whole-Exome Sequencing and Mass Spectrometry.

Background: Identifying genetic variation associated with plasma protein levels, and the mechanisms by which they act, could provide insight into alterable processes involved in regulation of protein levels. Although protein levels can be affected by genetic variants, their estimation can also be biased by missense variants in coding exons causing technical artifacts. Integrating genome sequence genotype data with mass spectrometry-based protein level estimation could reduce bias, thereby improving detection of variation that affects RNA or protein metabolism.

Dysregulated Microbial Fermentation of Soluble Fiber Induces Cholestatic Liver Cancer.

Dietary soluble fibers are fermented by gut bacteria into short-chain fatty acids (SCFA), which are considered broadly health-promoting. Accordingly, consumption of such fibers ameliorates metabolic syndrome. However, incorporating soluble fiber inulin, but not insoluble fiber, into a compositionally defined diet, induced icteric hepatocellular carcinoma (HCC).

Correction to: Mitochondrial H-ATP synthase in human skeletal muscle: contribution to dyslipidaemia and insulin resistance.

Owing to an oversight, the authors omitted to note that Dr Taub is a co-founder of and equity holder in Cardero Therapeutics.

Group B Streptococcus Biofilm Regulatory Protein A Contributes to Bacterial Physiology and Innate Immune Resistance.

Background: Streptococcus agalactiae (group B Streptococcus [GBS]) asymptomatically colonizes approximately 20% of adults; however, GBS causes severe disease in susceptible populations, including newborns, pregnant women, and elderly individuals. In shifting between commensal and pathogenic states, GBS reveals multiple mechanisms of virulence factor control. Here we describe a GBS protein that we named "biofilm regulatory protein A" (BrpA) on the basis of its homology with BrpA from Streptococcus mutans.

Defining Host Responses during Systemic Bacterial Infection through Construction of a Murine Organ Proteome Atlas.

Group A Streptococcus (GAS) remains one of the top 10 deadliest human pathogens worldwide despite its sensitivity to penicillin. Although the most common GAS infection is pharyngitis (strep throat), it also causes life-threatening systemic infections. A series of complex networks between host and pathogen drive invasive infections, which have not been comprehensively mapped.

Membrane-Derived Vesicles Promote Bacterial Virulence and Confer Protective Immunity in Murine Infection Models.

produces membrane-derived vesicles (MVs), which share functional properties to outer membrane vesicles. Atomic force microscopy revealed that -derived MVs are associated with the bacterial surface or released into the surrounding environment depending on bacterial growth conditions. By using a comparative proteomic approach, a total of 131 and 617 proteins were identified in MVs isolated from grown in Luria-Bertani and brain-heart infusion broth, respectively.

Identification of the S-transferase like superfamily bacillithiol transferases encoded by Bacillus subtilis.

Bacillithiol is a low molecular weight thiol found in Firmicutes that is analogous to glutathione, which is absent in these bacteria. Bacillithiol transferases catalyze the transfer of bacillithiol to various substrates. The S-transferase-like (STL) superfamily contains over 30,000 putative members, including bacillithiol transferases.

Biomimetic Virulomics for Capture and Identification of Cell-Type Specific Effector Proteins.

An unmet challenge in the study of disease is to accurately streamline the identification of important virulence factors. Traditional, genetically driven approaches miss biologically relevant markers due to discordance between the genome and proteome. Here, we developed a nanotechnology-enabled affinity enrichment strategy coupled with multiplexed quantitative proteomics, namely Biomimetic Virulomics, for successful identification of cell-type specific effector proteins of both prokaryotic and eukaryotic pathogens.

Detection of dysregulated protein-association networks by high-throughput proteomics predicts cancer vulnerabilities.

The formation of protein complexes and the co-regulation of the cellular concentrations of proteins are essential mechanisms for cellular signaling and for maintaining homeostasis. Here we use isobaric-labeling multiplexed proteomics to analyze protein co-regulation and show that this allows the identification of protein-protein associations with high accuracy. We apply this 'interactome mapping by high-throughput quantitative proteome analysis' (IMAHP) method to a panel of 41 breast cancer cell lines and show that deviations of the observed protein co-regulations in specific cell lines from the consensus network affects cellular fitness.

Mitochondrial H-ATP synthase in human skeletal muscle: contribution to dyslipidaemia and insulin resistance.

Aims/hypothesis: Mitochondria are important regulators of the metabolic phenotype in type 2 diabetes. A key factor in mitochondrial physiology is the H-ATP synthase. The expression and activity of its physiological inhibitor, ATPase inhibitory factor 1 (IF1), controls tissue homeostasis, metabolic reprogramming and signalling.

Quantitative Temporal Viromics of an Inducible HIV-1 Model Yields Insight to Global Host Targets and Phospho-Dynamics Associated with Protein Vpr.

The mechanisms by which human immunodeficiency virus (HIV) circumvents and coopts cellular machinery to replicate and persist in cells are not fully understood. HIV accessory proteins play key roles in the HIV life cycle by altering host pathways that are often dependent on post-translational modifications (PTMs). Thus, the identification of HIV accessory protein host targets and their PTM status is critical to fully understand how HIV invades, avoids detection and replicates to spread infection.

Proteomic analysis of pRb loss highlights a signature of decreased mitochondrial oxidative phosphorylation.

The retinoblastoma tumor suppressor (pRb) protein associates with chromatin and regulates gene expression. Numerous studies have identified Rb-dependent RNA signatures, but the proteomic effects of Rb loss are largely unexplored. We acutely ablated Rb in adult mice and conducted a quantitative analysis of RNA and proteomic changes in the colon and lungs, where Rb(KO) was sufficient or insufficient to induce ectopic proliferation, respectively.

Evidence of histidine and aspartic acid phosphorylation in human prostate cancer cells.

We have developed a method to identify previously undetected histidine and aspartic acid phosphorylations in a human prostate cancer progression model. A phosphoproteome of our cell line model is presented, with correlation of modified protein expression between the three states of cancer: non-tumorigenic, tumorigenic, and metastatic cells. With the described interaction proteins potentially phosphorylated by NM23-H1, cellular responses to motility and conformational change stimuli would be achievable.

Pseudomonas aeruginosa PA1006 is a persulfide-modified protein that is critical for molybdenum homeostasis.

A companion manuscript revealed that deletion of the Pseudomonas aeruginosa (Pae) PA1006 gene caused pleiotropic defects in metabolism including a loss of all nitrate reductase activities, biofilm maturation, and virulence. Herein, several complementary approaches indicate that PA1006 protein serves as a persulfide-modified protein that is critical for molybdenum homeostasis in Pae. Mutation of a highly conserved Cys22 to Ala or Ser resulted in a loss of PA1006 activity.

Proteomic and functional analyses of protein-DNA complexes during gene transfer.

One of the barriers to successful nonviral gene delivery is the crowded cytoplasm, which plasmids need to actively traverse for gene expression. Relatively little is known about how this process occurs, but our lab and others have shown that the microtubule network and motors are required for plasmid movement to the nucleus. To further investigate how plasmids exploit normal physiological processes to transfect cells, we have taken a proteomics approach to identify the proteins that comprise the plasmid-trafficking complex.

Cardiolipin biosynthesis in Streptococcus mutans is regulated in response to external pH.

Streptococcus mutans, a causative agent of dental caries in humans, adapts to changing environmental conditions, such as pH, in order to survive and cause disease in the oral cavity. Previously, we have shown that S. mutans increases the proportion of monounsaturated membrane fatty acids as part of its acid-adaptive strategy.

LC/LC-MS/MS of an innovative prostate human epithelial cancer (PHEC) in vitro model system.

This work describes the proteomic characterization of a novel in vitro prostate cancer model system, the clonal prostatic human epithelial cancer (PHEC) cell lines. The model is composed of three cell lines representing the three progressive cancer states found in vivo: non-tumorigenic, tumorigenic, and metastatic. The cell lines were evaluated for differential protein expression between states using two dimensional liquid:liquid chromatographic separation followed by mass spectral identification.

Mitogen- and stress-activated kinase 1 (MSK1) regulates cigarette smoke-induced histone modifications on NF-κB-dependent genes.

Cigarette smoke (CS) causes sustained lung inflammation, which is an important event in the pathogenesis of chronic obstructive pulmonary disease (COPD). We have previously reported that IKKα (I kappaB kinase alpha) plays a key role in CS-induced pro-inflammatory gene transcription by chromatin modifications; however, the underlying role of downstream signaling kinase is not known. Mitogen- and stress-activated kinase 1 (MSK1) serves as a specific downstream NF-κB RelA/p65 kinase, mediating transcriptional activation of NF-κB-dependent pro-inflammatory genes.

Mass spectrometry detection of histidine phosphorylation on NM23-H1.

Phosphorylation is a ubiquitous protein post-translational modification that is intimately involved in most aspects of cellular regulation. Currently, most proteomic analyses are performed with phosphorylation searches for serine, threonine, and tyrosine modifications, as the phosphorylated residues of histidine and aspartic acid are acid labile and thus undetectable with most proteomic methodologies. Here, we present a novel buffer system to show histidine phosphorylation of NM23-H1, the product of the first identified putative human metastasis suppressor gene (NME1), which catalyzes the transfer of the γ-phosphate from nucleoside triphosphates to nucleoside diphosphates.