Multifaceted Proteome Analysis at Solubility, Redox, and Expression Dimensions for Target Identification.

Multifaceted interrogation of the proteome deepens the system-wide understanding of biological systems; however, mapping the redox changes in the proteome has so far been significantly more challenging than expression and solubility/stability analyses. Here, the first high-throughput redox proteomics approach integrated with expression analysis (REX) is devised and combined with the Proteome Integral Solubility Alteration (PISA) assay. The whole PISA-REX experiment with up to four biological replicates can be multiplexed into a single tandem mass tag TMTpro set. For benchmarking this compact tool, HCT116 cells treated with auranofin are analyzed, showing great improvement compared with previous studies. PISA-REX is then applied to study proteome remodeling upon stimulation of human monocytes by interferon α (IFN-α). Applying this tool to study the proteome changes in plasmacytoid dendritic cells (pDCs) isolated from wild-type versus Ncf1-mutant mice treated with interferon α, shows that NCF1 deficiency enhances the STAT1 pathway and modulates the expression, solubility, and redox state of interferon-induced proteins. Providing comprehensive multifaceted information on the proteome, the compact PISA-REX has the potential to become an industry standard in proteomics and to open new windows into the biology of health and disease.

Gel-Assisted Proteome Position Integral Shift Assay Returns Molecular Weight to Shotgun Proteomics and Identifies Caspase 3 Substrates.

Here, we present a high-throughput virtual top-down proteomics approach that restores the molecular weight (MW) information in shotgun proteomics and demonstrates its utility in studying proteolytic events in programmed cell death. With gel-assisted proteome position integral shift (GAPPIS), we quantified over 7000 proteins in staurosporine-induced apoptotic HeLa cells and identified 84 proteins exhibiting in a statistically significant manner at least two of the following features: (i) a negative MW shift; (ii) an elevated ratio in a pair of a semitryptic and tryptic peptide, (iii) a negative shift in the standard deviation of MW estimated for different peptides, and (iv) a negative shift in skewness of the same data. Of these proteins, 58 molecules were previously unreported caspase 3 substrates. Further analysis identified the preferred cleavage sites consistent with the known caspase cleavages after the DXXD motif. As a powerful tool for high-throughput MW analysis simultaneously with the conventional expression analysis, the GAPPIS assay can prove useful in studying a broad range of biological processes involving proteolytic events.

Chemical Proteomics Reveals that the Anticancer Drug Everolimus Affects the Ubiquitin-Proteasome System.

Rapamycin is a natural antifungal, immunosuppressive, and antiproliferative compound that allosterically inhibits mTOR complex 1. The ubiquitin-proteasome system (UPS) responsible for protein turnover is usually not listed among the pathways affected by mTOR signaling. However, some previous studies have indicated the interplay between the UPS and mTOR. It has also been reported that rapamycin and its analogs can allosterically inhibit the proteasome itself. In this work, we studied the molecular effect of rapamycin and its analogs (rapalogs), everolimus and temsirolimus, on the A549 cell line by expression proteomics. The analysis of differentially expressed proteins showed that the cellular response to everolimus treatment is strikingly different from that to rapamycin and temsirolimus. In the cluster analysis, the effect of everolimus was similar to that of bortezomib, a well-established proteasome inhibitor. UPS-related pathways were enriched in the cluster of proteins specifically upregulated upon everolimus and bortezomib treatments, suggesting that both compounds have similar proteasome inhibition effects. In particular, the total amount of ubiquitin was significantly elevated in the samples treated with everolimus and bortezomib, and analysis of the polyubiquitination patterns revealed elevated intensities of the ubiquitin peptide with a GG modification at the K48 residue, consistent with a bottleneck in proteasomal protein degradation. Moreover, the everolimus treatment resulted in both ubiquitin phosphorylation and generation of a significant amount of semitryptic peptides, illustrating the increase in the protease activity. These observations suggest that everolimus affects the UPS in a unique way, and its mechanism of action is different from that of its close chemical analogs, rapamycin and temsirolimus.

Ultralight Ultrafast Enzymes.

Inorganic materials depleted of heavy stable isotopes are known to deviate strongly in some physicochemical properties from their isotopically natural counterparts. Here we explored for the first time the effect of simultaneous depletion of the heavy carbon, hydrogen, oxygen and nitrogen isotopes on the bacterium E. coli and the enzymes expressed in it. Bacteria showed faster growth, with most proteins exhibiting higher thermal stability, while for recombinant enzymes expressed in depleted media, faster kinetics was discovered. At room temperature, luciferase, thioredoxin and dihydrofolate reductase and Pfu DNA polymerase showed up to a 250 % increase in activity compared to the native counterparts, with an additional ∼50 % increase at 10 °C. Diminished conformational and vibrational entropy is hypothesized to be the cause of the accelerated kinetics. Ultralight enzymes may find an application where extreme reaction rates are required.

Massive Solubility Changes in Neuronal Proteins upon Simulated Traumatic Brain Injury Reveal the Role of Shockwaves in Irreversible Damage.

We investigated the immediate molecular consequences of traumatic brain injuries (TBIs) using a novel proteomics approach. We simulated TBIs using an innovative laboratory apparatus that employed a 5.1 kg dummy head that held neuronal cells and generated a ≤4000 g-force acceleration upon impact. A Proteome Integral Solubility Alteration (PISA) assay was then employed to monitor protein solubility changes in a system-wide manner. Dynamic impacts led to both a reduction in neuron viability and massive solubility changes in the proteome. The affected proteins mapped not only to the expected pathways, such as those of cell adhesion, collagen, and laminin structures, as well as the response to stress, but also to other dense protein networks, such as immune response, complement, and coagulation cascades. The cellular effects were found to be mainly due to the shockwave rather than the g-force acceleration. Soft materials could reduce the impact's severity only until they were fully compressed. This study shows a way of developing a proteome-based meter for measuring irreversible shockwave-induced cell damage and provides a resource for identifying protein biomarkers of TBIs and potential drug targets for the development of products aimed at primary prevention and intervention.

NCF1-dependent production of ROS protects against lupus by regulating plasmacytoid dendritic cell development and functions.

Low capacity to produce ROS because of mutations in neutrophil cytosolic factor 1 (NCF1/p47phox), a component of NADPH oxidase 2 (NOX2) complex, is strongly associated with systemic lupus erythematosus in both humans and mouse models. Here, we aimed to identify the key immune cell type(s) and cellular mechanisms driving lupus pathogenesis under the condition of NCF1-dependent ROS deficiency. Using cell-specific Cre-deleter, human NCF1-339 variant knockin, and transgenic mouse strains, we show that low ROS production in plasmacytoid dendritic cells (pDCs) exacerbated both pristane-induced lupus and a potentially new Y-linked autoimmune accelerating locus-related spontaneous model by promoting pDC accumulation in multiple organs during lupus development, accompanied by elevated IFN-α levels and expression of IFN-stimulated genes. Mechanistic studies revealed that ROS deficiency enhanced pDC generation through the AKT/mTOR pathway and CCR2-mediated migration to tissues, which together with hyperactivation of the redox-sensitive stimulator of interferon genes/IFN-α/JAK1/STAT1 cascade further augmented type I IFN responses. More importantly, by suppressing these pathways, restoration of NOX2-derived ROS specifically in pDCs protected against lupus. These discoveries explain the causative effect of dysfunctional NCF1 in lupus and demonstrate the protective role of pDC-derived ROS in disease development driven by NCF1-dependent ROS deficiency.