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. We hypothesized that reversible, lysine-targeted inhibitors could provide sustained kinase engagement in vivo, with selectivity driven in part by differences in residence time. By strategically linking benzaldehydes to a promiscuous kinase binding scaffold, we developed chemoproteomic probes that reversibly and covalently engage >200 protein kinases in cells and mice. Probe-kinase residence time was dramatically enhanced by a hydroxyl group ortho to the aldehyde. Remarkably, only a few kinases, including Aurora A, showed sustained, quasi-irreversible occupancy in vivo, the structural basis for which was revealed by X-ray crystallography. We anticipate broad application of salicylaldehyde-based probes to proteins that lack a druggable cysteine.

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. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal-regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal-regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.

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. Through single cell tracing of Cdk4i cells, we identified a time-sensitive early G1 phase specific Cdk4/6-dependent phosphorylation gradient that regulates cell cycle entry timing and resides between serum-sensing and cyclin E:Cdk2 activation. To reveal the substrate identity of the Cdk4/6 phosphorylation gradient, we performed whole proteomic and phospho-proteomic mass spectrometry, and identified 147 proteins and 82 phospho-peptides that significantly changed due to Cdk4 inhibition in early G1 phase. In summary, we identified novel (non-Rb) cyclin D:Cdk4/6 substrates that connects early G1 phase functions with cyclin E:Cdk2 activation and Rb inactivation by hyper-phosphorylation.

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. Despite progress in understanding PAR1 biased signaling, the proteins and pathways utilized by thrombin versus APC signaling to induce opposing cellular functions are largely unknown. Here, we report the global phosphoproteome induced by thrombin and APC signaling in endothelial cells with the quantification of 11,266 unique phosphopeptides using multiplexed quantitative mass spectrometry. Our results reveal unique dynamic phosphoproteome profiles of thrombin and APC signaling, an enrichment of associated biological functions, including key modulators of endothelial barrier function, regulators of gene transcription, and specific kinases predicted to mediate PAR1 biased signaling. Using small interfering RNA to deplete a subset of phosphorylated proteins not previously linked to thrombin or APC signaling, a function for afadin and adducin-1 actin binding proteins in thrombin-induced endothelial barrier disruption is unveiled. Afadin depletion resulted in enhanced thrombin-promoted barrier permeability, whereas adducin-1 depletion completely ablated thrombin-induced barrier disruption without compromising p38 signaling. However, loss of adducin-1 blocked APC-induced Akt signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 biased signaling in endothelial cells.

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). This assay was validated with papain, a well-characterized cysteine peptidase, to generate cleavage efficiency values for hydrolysis of 275 unique peptide bonds in parallel. To demonstrate the breath of this assay, we show that qMSP-MS can uncover the substrate specificity of minimally characterized intramembrane rhomboid peptidases, as well as define hundreds of proteolytic activities in complex biological samples, including secretions from lung cancer cell lines. Importantly, our qMSP-MS library uses synthetic peptides whose termini are unmodified, allowing us to characterize not only endo- but also exo-peptidase activity. Each cleaved peptide sequence can be ranked by turnover rate, and the amino acid sequence of the best substrates can be used for designing fluorescent reporter substrates. Discovery of peptide substrates that are selectively cleaved by peptidases which are active at the site of disease highlights the potential for qMSP-MS to guide the development of peptidase-activating drugs for cancer and infectious disease.

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, Mycoplasma mycoides capri, 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 0.59. The genes in the reconstruction have a high in vivo essentiality or quasi-essentiality of 92% (68% essential), compared to 79% in silico essentiality. This coherent model of the minimal metabolism in JCVI-syn3A at the same time also points toward specific open questions regarding the minimal genome of JCVI-syn3A, which still contains many genes of generic or completely unclear function. In particular, the model, its comparison to in vivo essentiality and proteomics data yield specific hypotheses on gene functions and metabolic capabilities; and provide suggestions for several further gene removals. In this way, the model and its accompanying data guide future investigations of the minimal cell. Finally, the identification of 30 essential genes with unclear function will motivate the search for new biological mechanisms beyond metabolism.

One way that researchers can test whether they understand a biological system is to see if they can accurately recreate it as a computer model. The more they learn about living things, the more the researchers can improve their models and the closer the models become to simulating the original. In this approach, it is best to start by trying to model a simple system. Biologists have previously succeeded in creating 'minimal bacterial cells'. These synthetic cells contain fewer genes than almost all other living things and they are believed to be among the simplest possible forms of life that can grow on their own. The minimal cells can produce all the chemicals that they need to survive ­ in other words, they have a metabolism. Accurately recreating one of these cells in a computer is a key first step towards simulating a complete living system. Breuer et al. have developed a computer model to simulate the network of the biochemical reactions going on inside a minimal cell with just 493 genes. By altering the parameters of their model and comparing the results to experimental data, Breuer et al. explored the accuracy of their model. Overall, the model reproduces experimental results, but it is not yet perfect. The differences between the model and the experiments suggest new questions and tests that could advance our understanding of biology. In particular, Breuer et al. identified 30 genes that are essential for life in these cells but that currently have no known purpose. Continuing to develop and expand models like these to reproduce more complex living systems provides a tool to test current knowledge of biology. These models may become so advanced that they could predict how living things will respond to changing situations. This would allow scientists to test ideas sooner and make much faster progress in understanding life on Earth. Ultimately, these models could one day help to accelerate medical and industrial processes to save lives and enhance productivity.

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. METHODS: Here, we integrate the blood plasma protein levels of 664 proteins from 165 participants of the Tromsø Study, measured via tandem mass tag mass spectrometry, with whole-exome sequencing data to identify common and rare genetic variation associated with peptide and protein levels (protein quantitative trait loci [pQTLs]). We additionally use literature and database searches to prioritize putative functional variants for each pQTL. RESULTS: We identify 109 independent associations (36 protein and 73 peptide) and use genotype data to exclude 49 (4 protein and 45 peptide) as technical artifacts. We describe 2 particular cases of rare variation: 1 associated with the complement pathway and 1 with platelet degranulation. We identify putative functional variants and show that pQTLs act through diverse molecular mechanisms that affect both RNA and protein metabolism. CONCLUSIONS: We show that although the majority of pQTLs exert their effects by modulating RNA metabolism, many affect protein levels directly. Our work demonstrates the extent by which pQTL studies are affected by technical artifacts and highlights how prioritizing the functional variant in pQTL studies can lead to insights into the molecular steps by which a protein may be regulated.

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.

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). Such HCC was microbiota-dependent and observed in multiple strains of dysbiotic mice but not in germ-free nor antibiotics-treated mice. Furthermore, consumption of an inulin-enriched high-fat diet induced both dysbiosis and HCC in wild-type (WT) mice. Inulin-induced HCC progressed via early onset of cholestasis, hepatocyte death, followed by neutrophilic inflammation in liver. Pharmacologic inhibition of fermentation or depletion of fermenting bacteria markedly reduced intestinal SCFA and prevented HCC. Intervening with cholestyramine to prevent reabsorption of bile acids also conferred protection against such HCC. Thus, its benefits notwithstanding, enrichment of foods with fermentable fiber should be approached with great caution as it may increase risk of HCC.

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. Methods: We coupled phenotypic assays, RNA sequencing, human neutrophil and whole-blood killing assays, and murine infection models to investigate the contribution of BrpA to GBS physiology and virulence. Results: Sequence analysis identified BrpA as a LytR-CpsA-Psr enzyme. Targeted mutagenesis yielded a GBS mutant (ΔbrpA) with normal ultrastructural morphology but a 6-fold increase in chain length, a biofilm defect, and decreased acid tolerance. GBS ΔbrpA stimulated increased neutrophil reactive oxygen species and proved more susceptible to human and murine blood and neutrophil killing. Notably, the wild-type parent outcompeted ΔbrpA GBS in murine sepsis and vaginal colonization models. RNA sequencing of ΔbrpA uncovered multiple differences from the wild-type parent, including pathways of cell wall synthesis and cellular metabolism. Conclusions: We propose that BrpA is an important virulence regulator and potential target for design of novel antibacterial therapeutics against GBS.

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. Attempting to map these interactions, we examined organ-level protein dynamics using a mouse model of systemic GAS infection. We quantified over 11,000 proteins, defining organ-specific markers for all analyzed tissues. From this analysis, an atlas of dynamically regulated proteins and pathways was constructed. Through statistical methods, we narrowed organ-specific markers of infection to 34 from the defined atlas. We show these markers are trackable in blood of infected mice, and a subset has been observed in plasma samples from GAS-infected clinical patients. This proteomics-based strategy provides insight into host defense responses, establishes potentially useful targets for therapeutic intervention, and presents biomarkers for determining affected organs during bacterial infection.

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

Staphylococcus aureus produces membrane-derived vesicles (MVs), which share functional properties to outer membrane vesicles. Atomic force microscopy revealed that S. aureus-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 S. aureus grown in Luria-Bertani and brain-heart infusion broth, respectively. Purified S. aureus MVs derived from the bacteria grown in either media induced comparable levels of cytotoxicity and neutrophil-activation. Administration of exogenous MVs increased the resistance of S. aureus to killing by whole blood or purified human neutrophils ex vivo and increased S. aureus survival in vivo. Finally, immunization of mice with S. aureus-derived MVs induced production of IgM, total IgG, IgG1, IgG2a, and IgG2b resulting in protection against subcutaneous and systemic S. aureus infection. Collectively, our results suggest S. aureus MVs can influence bacterial-host interactions during systemic infections and provide protective immunity in murine models of infection.

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. Proteins in this family are extremely divergent and are related by structural rather than sequence similarity, leaving it unclear if all share the same biochemical activity. Bacillus subtilis encodes eight predicted STL superfamily members, only one of which has been shown to be a bacillithiol transferase. Here we find that the seven remaining proteins show varying levels of metal dependent bacillithiol transferase activity. We have renamed the eight enzymes BstA-H. Mass spectrometry and gene expression studies revealed that all of the enzymes are produced to varying levels during growth and sporulation, with BstB and BstE being the most abundant and BstF and BstH being the least abundant. Interestingly, several bacillithiol transferases are induced in the mother cell during sporulation. A strain lacking all eight bacillithiol transferases showed normal growth in the presence of stressors that adversely affect growth of bacillithiol-deficient strains, such as paraquat and CdCl2. Thus, the STL bacillithiol transferases represent a new group of proteins that play currently unknown, but potentially significant roles in bacillithiol-dependent reactions. We conclude that these enzymes are highly divergent, perhaps to cope with an equally diverse array of endogenous or exogenous toxic metabolites and oxidants.

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. Furthermore, these aberrant interactions serve as biomarkers that predict the drug sensitivity of cell lines in screens across 195 drugs. We expect that IMAHP can be broadly used to gain insight into how changing landscapes of protein-protein associations affect the phenotype of biological systems.

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. We highlight the power of Biomimetic Virulomics by capturing known virulence factors in a high-throughput, cell-type guided fashion. Additionally, a comprehensive characterization of the membrane protein component of biomimetics utilized in this strategy is provided. Interfacing cell-derived nanomaterials with multiplexed quantitative proteomics allow for a specific targeting strategy of virulence factors that can be utilized for drug discovery against prominent human diseases.

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. We aimed to characterise the putative role of IF1 in mediating skeletal muscle metabolism in obesity and diabetes. METHODS: We examined the 'mitochondrial signature' of obesity and type 2 diabetes in a cohort of 100 metabolically characterised human skeletal muscle biopsy samples. The expression and activity of H+-ATP synthase, IF1 and key mitochondrial proteins were characterised, including their association with BMI, fasting plasma insulin, fasting plasma glucose and HOMA-IR. IF1 was also overexpressed in primary cultures of human myotubes derived from the same biopsies to unveil the possible role played by the pathological inhibition of the H+-ATP synthase in skeletal muscle. RESULTS: The results indicate that type 2 diabetes and obesity act via different mechanisms to impair H+-ATP synthase activity in human skeletal muscle (76% reduction in its catalytic subunit vs 280% increase in IF1 expression, respectively) and unveil a new pathway by which IF1 influences lipid metabolism. Mechanistically, IF1 altered cellular levels of α-ketoglutarate and L-carnitine metabolism in the myotubes of obese (84% of control) and diabetic (76% of control) individuals, leading to limited ß-oxidation of fatty acids (60% of control) and their cytosolic accumulation (164% of control). These events led to enhanced release of TNF-α (10 ± 2 pg/ml, 27 ± 5 pg/ml and 35 ± 4 pg/ml in control, obese and type 2 diabetic participants, respectively), which probably contributes to an insulin resistant phenotype. CONCLUSIONS/INTERPRETATION: Overall, our data highlight IF1 as a novel regulator of lipid metabolism and metabolic disorders, and a possible target for therapeutic intervention.

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. To date, a comprehensive characterization of HIV accessory protein host targets and modulation of their PTM status does not exist. The significant gap in knowledge regarding the identity and PTMs of HIV host targets is due, in part, to technological limitations. Here, we applied current mass spectrometry techniques to define mechanisms of viral protein action by identifying host proteins whose abundance is affected by the accessory protein Vpr and the corresponding modulation of down-stream signaling pathways, specifically those regulated by phosphorylation. By utilizing a novel, inducible HIV-1 CD4+ T-cell model system expressing either the wild type or a vpr-negative viral genome, we overcame challenges associated with synchronization and infection-levels present in other models. We report identification and abundance dynamics of over 7000 proteins and 28,000 phospho-peptides. Consistent with Vpr's ability to impair cell-cycle progression, we observed Vpr-mediated modulation of spindle and centromere proteins, as well as Aurora kinase A and cyclin-dependent kinase 4 (CDK4). Unexpectedly, we observed evidence of Vpr-mediated modulation of the activity of serine/arginine-rich protein-specific kinases (SRPKs), suggesting a possible role for Vpr in the regulation of RNA splicing. This study presents a new experimental system and provides a data-resource that lays the foundation for validating host proteins and phosphorylation-pathways affected by HIV-1 and its accessory protein Vpr.

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. As expected, Rb(KO) caused similar increases in classic pRb/E2F-regulated transcripts in both tissues, but, unexpectedly, their protein products increased only in the colon, consistent with its increased proliferative index. Thus, these protein changes induced by Rb loss are coupled with proliferation but uncoupled from transcription. The proteomic changes in common between Rb(KO) tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and oxidative phosphorylation (OXPHOS) function. RB(KO) cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb loss altered mitochondrial pyruvate oxidation from (13)C-glucose through the TCA cycle in mouse tissues and cultured cells. Consequently, RB(KO) cells have an enhanced sensitivity to mitochondrial stress conditions. In summary, proteomic analyses provide a new perspective on Rb/RB1 mutation, highlighting the importance of pRb for mitochondrial function and suggesting vulnerabilities for treatment.

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. We detect 20 novel histidine-phosphorylated (pHis) and 80 novel aspartic acid-phosphorylated (pAsp) proteins with diverse functions, such as metabolism, protein folding, and motility. Our data indicate that pHis and pAsp are much more prevalent than previously appreciated and may provide insight into the role of NM23-H1 and signaling events that are critical for metastasis. Using the described method for detecting histidine and aspartic acid phosphorylations and our prostate cancer progression cell system, the potential function of NM23-H1 in suppressing metastasis with a two-component regulation system is discussed.

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. Yeast-two-hybrid and a green-fluorescent protein fragment complementation assay (GFP-PFCA) in Pae itself revealed that PA1006 interacts with Pae PA3667/CsdA and PA3814/IscS Cys desulfurase enzymes. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) "top-down" analysis of PA1006 purified from Pae revealed that conserved Cys22 is post-translationally modified in vivo in the form a persulfide. Inductively-coupled-plasma (ICP)-MS analysis of ΔPA1006 mutant extracts revealed that the mutant cells contain significantly reduced levels of molybdenum compared to wild-type. GFP-PFCA also revealed that PA1006 interacts with several molybdenum cofactor (MoCo) biosynthesis proteins as well as nitrate reductase maturation factor NarJ and component NarH. These data indicate that a loss of PA1006 protein's persulfide sulfur and a reduced availability of molybdenum contribute to the phenotype of a ΔPA1006 mutant.