Global Phosphoproteomic Analysis Research Articles

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.

Global Phosphoproteomics Analysis of IBRS-2 Cells Infected With Senecavirus A.

Phosphorylation is a widespread posttranslational modification that regulates numerous biological processes. Viruses can alter the physiological activities of host cells to promote virus particle replication, and manipulating phosphorylation is one of the mechanisms. Senecavirus A (SVA) is the causative agent of porcine idiopathic vesicular disease. Although numerous studies on SVA have been performed, comprehensive phosphoproteomics analysis of SVA infection is lacking. The present study performed a quantitative mass spectrometry-based phosphoproteomics survey of SVA infection in Instituto Biologico-Rim Suino-2 (IBRS-2) cells. Three parallel experiments were performed, and 4,520 phosphosites were quantified on 2,084 proteins. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that many phosphorylated proteins were involved in apoptosis and spliceosome pathways, and subcellular structure localization analysis revealed that more than half were located in the nucleus. Motif analysis of proteins with differentially regulated phosphosites showed that proline, aspartic acid, and glutamic acid were the most abundant residues in the serine motif, while proline and arginine were the most abundant in the threonine motif. Forty phosphosites on 27 proteins were validated by parallel reaction monitoring (PRM) phosphoproteomics, and 30 phosphosites in 21 proteins were verified. Nine proteins with significantly altered phosphosites were further discussed, and eight [SRRM2, CDK13, DDX20, DDX21, BAD, ELAVL1, PDZ-binding kinase (PBK), and STAT3] may play a role in SVA infection. Finally, kinase activity prediction showed 10 kinases’ activity was reversed following SVA infection. It is the first phosphoproteomics analysis of SVA infection of IBRS-2 cells, and the results greatly expand our knowledge of SVA infection. The findings provide a basis for studying the interactions of other picornaviruses and their mammalian host cells.

A global phosphoproteomics analysis of adult Fasciola gigantica by LC-MS/MS.

Protein phosphorylation plays key roles in a variety of essential cellular processes. Fasciola gigantica is a tropical liver fluke causing hepatobiliary disease fascioliasis, leading to human health threats and heavy economic losses. Although the genome and protein kinases of F. gigantica provided new insights to understand the molecular biology and etiology of this parasite, there is scant knowledge of protein phosphorylation events in F. gigantica. In this study, we characterized the global phosphoproteomics of adult F. gigantica by phosphopeptide enrichment-based LC–MS/MS, a high-throughput analysis to maximize the detection of a large repertoire of phosphoproteins and phosphosites. A total of 1030 phosphopeptides with 1244 phosphosites representing 635 F. gigantica phosphoproteins were identified. The phosphoproteins were involved in a wide variety of biological processes including cellular, metabolic, and single-organism processes. Meanwhile, these proteins were found predominantly in cellular components like membranes and organelles with molecular functions of binding (51.3%) and catalytic activity (40.6%). The KEGG annotation inferred that the most enriched pathways of the phosphoproteins included tight junction, spliceosome, and RNA transport (each one contains 15 identified proteins). Combining the reports in other protozoa and helminths, the phosphoproteins identified in this work play roles in metabolic regulation and signal transduction. To our knowledge, this work performed the first global phosphoproteomics analysis of adult F. gigantica, which provides valuable information for development of intervention strategies for fascioliasis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00436-021-07422-2.

Abstract P071: Phosphoproteomics identifies Mig6 as a key mediator of adaptive resistance to ALK/ROS1 oncogene inhibition

Abstract Tyrosine kinase inhibitors (TKIs) targeting ALK and ROS1 fusions have significantly improved outcomes for patients with non-small cell lung cancer harboring these oncogenes. However, complete responses are rarely achieved, and therapeutic resistance eventually emerges from residual tumor. To control or eradicate residual disease pre-emptively, it is important to understand the molecular mechanism contributing to the adaptive resistance under initial exposure to oncogene inhibition. Our lab previously demonstrated that EGFR mediates responses to TKIs in oncogene fusion positive cancers, but the detailed mechanism underlying adaptive EGFR signaling is still not clear. We performed global and tyrosine-specific phosphoproteomic analyses to investigate the signaling reprogramming following 2-hr treatment of crizotinib, a dual ALK/ROS1 inhibitor, in an EML4-ALK cell line (H3122) and a TPM3-ROS1 patient-derived cell line (CUTO28). Pathway analysis revealed “ErbB signaling pathway” and “regulation of ErbB signaling pathway” were enriched in the proteins with differentially expressed phosphosites under crizotinib treatment. Mig6, a protein that negatively regulates EGFR, showed a significant decrease of phosphorylation of Y394/Y395 following crizotinib treatment. Phosphorylation of these 2 residues has been shown to be essential for Mig6 binding and subsequent inhibition of EGFR, suggesting that EGFR activity may be released from Mig6 inhibition rapidly following ALK or ROS1 inhibition. Mig6 transcripts and protein levels were also reduced by following ALK/ROS1 inhibition. The MEK inhibitor trametinib decreased Mig6 transcripts and protein with a similar magnitude as ALK/ROS1 inhibition, suggesting that the regulation of Mig6 expression by ALK/ROS1 inhibition is mediated by the MAPK pathway. Mig6 knock-down was able to rescue cell survival and p-Erk suppression from ALK/ROS1 inhibition, but failed to rescue when co-treated with afatinib, a pan-HER inhibitor. Neither Mig6 knockdown nor overexpression altered the phosphorylation of EGFR itself. Instead, phosphorylation of Y239 and Y240 on Shc1, a signaling adapter shared by ALK, ROS1, and EGFR, was rescued by Mig6 knockdown under ALK/ROS1 inhibition. This is consistent with the previously reported structures showing Mig6 and Shc1 compete for the same substrate binding cleft on EGFR. Combination of afatinib and ALK/ROS1 TKIs eliminated residual colony formation in 2 weeks. Crizotinib-resistant H3122 demonstrated loss of Mig6 and resistance could be reversed using either afatinib or Mig6 overexpression. In summary, this study identified Mig6 as a novel regulator for ErbB signaling-mediated adaptive and acquired resistance to ALK/ROS1 TKIs. This resistance mechanism may have broader implications for other oncogene-driven cancers given the observed Mig6 regulation by the MAPK pathway. These data provide additional support for co-inhibition of EGFR in oncogene-driven cancers, such as ALK or ROS1, to reduce the burden of residual disease by eliminating Mig6-mediated EGFR survival pathways. Citation Format: Nan Chen, Anh T. Le, Eric A. Welsh, Bin Fang, Eric B. Haura, Robert C. Doebele. Phosphoproteomics identifies Mig6 as a key mediator of adaptive resistance to ALK/ROS1 oncogene inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P071.

Cholesterol Regulates the Tumor Adaptive Resistance to MAPK Pathway Inhibition.

Although targeted MAPK pathway inhibition has achieved remarkable patient responses in many cancers, the development of resistance has remained a critical challenge. Adaptive tumor response underlies the drug resistance. Furthermore, such bypass mechanisms often lead to the activation of many pro-survival kinases, which complicates the rational design of combination therapies. Here, we performed global tyrosine phosphoproteomic (pTyr) analyses and demonstrated that targeted MAPK signaling inhibition in melanoma leads to a profound remodeling of the pTyr proteome. Intriguingly, altered cholesterol metabolism might drive, in a coordinated fashion, the activation of these kinases. Indeed, we found an accumulation of intracellular cholesterol in melanoma cells (with BRAFV600E mutations) and non-small cell lung cancer cells (with KRASG12C mutations) treated with MAPK and KRASG12C inhibitors, respectively. Importantly, depletion of cholesterol not only prevents the feedback activation of pTyr signaling but also enhances the cytotoxic effects of MAPK pathway inhibitors, both in vitro and in vivo. Together, our findings suggest that cholesterol contributes to the tumor adaptive response upon targeted MAPK pathway inhibitors. These results also suggest that MAPK pathway inhibitors could be combined with cholesterol-lowering agents to achieve a more complete and durable response in tumors with hyperactive MAPK signaling.

Global phosphoproteome analysis reveals significant differences between sporulated oocysts of virulent and avirulent strains of Toxoplasma gondii

In this study, the differences in the phosphoproteomic landscape of sporulated oocysts between virulent and avirulent strains of Toxoplasma gondii were examined using a global phosphoproteomics approach. Phosphopeptides from sporulated oocysts of the virulent PYS strain (Chinese ToxoDB#9) and the avirulent PRU strain (type II) were enriched by titanium dioxide (TiO2) affinity chromatography and quantified using IBT approach. A total of 10,645 unique phosphopeptides, 8181 nonredundant phosphorylation sites and 2792 phosphoproteins were identified. We also detected 4129 differentially expressed phosphopeptides (DEPs) between sporulated oocysts of PYS strain and PRU strain (|log1.5 fold change| > 1 and p < 0.05), including 2485 upregulated and 1644 downregulated phosphopeptides. Motif analysis identified 24 motifs from the upregulated phosphorylated peptides including 22 serine motifs and two threonine motifs (TPE and TP), and 15 motifs from the downregulated phosphorylated peptides including 12 serine motifs and three threonine motifs (TP, RxxT and KxxT) in PYS strain when comparing PYS strain to PRU strain. Several kinases were consistent with motifs of overrepresented phosphopeptides, such as PKA, PKG, CKII, IKK, MAPK, EGFR, INSR, Jak, Syk, Src, Ab1. GO enrichment, KEGG pathway analysis and STRING analysis revealed DEPs significantly enriched in many biological processes and pathways. Kinase related network analysis showed that AGC kinase was the most connected kinase peptide. Our findings reveal significant difference in phosphopeptide profiles of sporulated oocysts between virulent and avirulent T. gondii strains, providing new resources for further elucidation of the mechanisms underpinning the virulence of T. gondii.

Inositol polyphosphates and target of rapamycin kinase signalling govern photosystem II protein phosphorylation and photosynthetic function under light stress in Chlamydomonas

Stress and nutrient availability influence cell proliferation through complex intracellular signalling networks. In a previous study it was found that pyro-inositol polyphosphates (InsP7 and InsP8 ) produced by VIP1 kinase, and target of rapamycin (TOR) kinase signalling interacted synergistically to control cell growth and lipid metabolism in the green alga Chlamydomonas reinhardtii. However, the relationship between InsPs and TOR was not completely elucidated. We used an invivo assay for TOR activity together with global proteomic and phosphoproteomic analyses to assess differences between wild-type and vip1-1 in the presence and absence of rapamycin. We found that TOR signalling is more severely affected by the inhibitor rapamycin in a vip1-1 mutant compared with wild-type, indicating that InsP7 and InsP8 produced by VIP1 act independently but also coordinately with TOR. Additionally, among hundreds of differentially phosphorylated peptides detected, an enrichment for photosynthesis-related proteins was observed, particularly photosystem II proteins. The significance of these results was underscored by the finding that vip1-1 strains show multiple defects in photosynthetic physiology that were exacerbated under high light conditions. These results suggest a novel role for inositol pyrophosphates and TOR signalling in coordinating photosystem phosphorylation patterns in Chlamydomonas cells in response to light stress and possibly other stresses.

Quantitative Proteomics and Phosphoproteomics Reveal TNF-α-Mediated Protein Functions in Hepatocytes.

Increased secretion of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNFα), is often associated with adipose tissue dysregulation, which often accompanies obesity. High levels of TNFα have been linked to the development of insulin resistance in several tissues and organs, including skeletal muscle and the liver. In this study, we examined the complex regulatory roles of TNFα in murine hepatocytes utilizing a combination of global proteomic and phosphoproteomic analyses. Our results show that TNFα promotes extensive changes not only of protein levels, but also the dynamics of their downstream phosphorylation signaling. We provide evidence that TNFα induces DNA replication and promotes G1/S transition through activation of the MAPK pathway. Our data also highlight several other novel proteins, many of which are regulated by phosphorylation and play a role in the progression and development of insulin resistance in hepatocytes.

Phosphoproteomic Characterization of Primary AML Samples and Relevance for Response Toward FLT3-inhibitors.

Phosphoproteomic Characterization of Primary AML Samples and Relevance for Response Toward FLT3-inhibitors.

Abstract 2172: Proteogenomics of laser microdissected difficult-to-treat breast cancers identifies Basal and Her2 patients associated with outcome differences

Abstract Introduction: Breast cancer (BC) is the second most common cause of cancer death among women in the USA. Recent proteogenomic studies of human BC have identified many potential therapeutic biomarkers for the most common types of BC. Here, we strive to comprehensively understand the proteogenomic landscape of difficult-to-treat breast cancer (DTBC) tumors. Methods: 118 primary breast tumors, with a focus on DTBC patients, were selected from the IRB-approved protocol. The study cohort included 30 triple negative, 16 HER2+, 16 Luminal A, 39 Luminal B1, and 17 Luminal B2 selected by immunohistochemistry. Breast tumors were embedded in OCT (Optimum Cutting Temperature) and processed by laser microdissection (LMD) to remove non-tumorous tissue. DNA, RNA, and protein were simultaneously extracted from each tumor using the illustra triplePrep kit. Paired-end RNA sequencing and whole genome sequencing (WGS) were performed for 118 and 100 tumors, respectively, using the Illumina HiSeq platform. Quantitative global proteomics and phosphoproteomic analyses were performed on 113 and 50 tumors, respectively, using isobaric TMT 6-plex labeling with the “universal reference” strategy. Results: Unlike other BC cohorts, our WGS cohort had an almost equal number of Estrogen(ER)+ (55.6%) and ER- (44.4%) cases, which gave us a nominally higher percentage TP53 (57%) and lower PIK3CA (32%) non-silent somatic mutation. Similar to previous reports, GATA3 was mutated in 13% of only ER+ cases. Unlike a recent report on proteomics clustering of bulk-processed tumors, our data show that a stromal-enriched cluster was not mixed with all subtypes but a majority of Luminal A (LumA) subtype, probably because LMD excluded stromal components in other tumor subtypes. Consensus clustering of proteomics data separated intrinsic Her2 patients with outcome differences. Her2 patients associated with the Basal-enriched proteome cluster had better survival than those associated with the LumA-enriched proteome cluster. Previously reported marker PGK1, a predictor for poor survival in BC, was upregulated in Her2 tumors associated with the LumA-enriched proteome cluster. The clustering of phosphoproteomics data separated intrinsic Basal patients into groups with and without relapse. We identified 3 and 18 genes with upregulated kinase activities in the relapse-free and relapsed Basal patients, respectively. In conclusion, this study provides a high-quality proteogenomic resource for DTBC investigation and identifies potential molecular markers for predicting outcomes for patients with less responsive tumors. The views expressed in this abstract are solely of the authors and do not reflect the official policy of the Departments of Army/Navy/Air Force, DoD, USUHS, HJF. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Citation Format: Praveen-Kumar Raj-Kumar, Tao Liu, Lori A. Sturtz, Albert J. Kovatich, Marina A. Gritsenko, Vladislav A. Petyuk, Brenda Deyarmin, Jianfang Liu, Anupama Praveen-Kumar, Jason E. McDermott, Anil K. Shukla, Ronald J. Moore, Matthew E. Monroe, Bobbie-Jo M. Webb-Robertson, Jeffrey A. Hooke, Leigh Fantacone-Campbell, Leonid Kvecher, Jennifer Kane, Jennifer Melley, Stella Somiari, Stephen C. Benz, Justin Golovato, Shahrooz Rabizadeh, Patrick Soon-Shiong, Richard D. Smith, Richard J. Mural, Karin D. Rodland, Craig D. Shriver, Zhaoyu Li, Hai Hu. Proteogenomics of laser microdissected difficult-to-treat breast cancers identifies Basal and Her2 patients associated with outcome differences [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2172.

Sustained Reduction of Intracellular Calcium Is Important to Maximise GA101-Mediated Direct Cell Death in B-Cell Lymphomas

For over a decade, anti-CD20 monoclonal antibodies (mAbs) have successfully been used in the therapy of B-cell malignancies. Obinutuzumab (GA101), a type-II glycoengineered anti-CD20 mAb approved for treatment of Chronic Lymphocytic Leukaemia (CLL) and Follicular Lymphoma, was designed to have enhanced antibody-dependant cellular cytotoxicity compared with Rituximab. Pre-clinical data showed that in addition, GA101 also exhibited significantly enhanced direct cell death. The complete mechanism of increased direct cell death is still unknown.The biological role of CD20 remains unclear but it has been shown to act as part of an ion channel complex, a component of the store operated calcium (Ca2+) entry system. Numerous studies have investigated the immediate impact of anti-CD20 mAbs on intracellular Ca2+. Yet to date, none have assessed intracellular Ca2+ over a longer period.To investigate the effect of anti-CD20 mAbs on intracellular Ca2+, we initially used 4 B-cell lymphoma cell lines to assess Ca2+ flux immediately after treatment with either Rituximab or GA101. Cells were first stained with a fluorescent calcium indicator and baseline measurements recorded by flow cytometry. Cells were then treated with each antibody and fluorescence measurements were recorded for a further 5-10 minutes. Next, we conducted a time course experiment to visualise the kinetics of intracellular Ca2+ after 1, 2, 4, 8, and 24 hour treatments with either Rituximab or GA101; Trastuzumab was used as an isotype control. After treatment, samples were stained with a labelled calcium indicator before quantifying intracellular Ca2+ using imaging flow cytometry. We validated our findings in primary CLL samples using the same method. We conducted liquid chromatography tandem mass spectrometry based label-free phosphoproteomics on SUDHL4 cells in biological triplicates after 1 and 24 hour treatments with GA101 and Rituximab. Expression levels of >8500 phosphorylation sites were measured across treatments to establish changes in intracellular signalling and kinase activity.In line with published data, we observed an influx of Ca2+ when cells were treated with Rituximab but intracellular Ca2+ was not significantly altered for any of the extended treatments. In contrast, GA101 treatment did not induce an immediate influx of Ca2+,and a reduction in intracellular Ca2+ was observed after 1 hour treatment which was sustained across all extended treatment periods. This difference in kinetics was validated in primary CLL samples. Global phosphoproteomic analysis found that the activity of Polo-Like Kinase 2 (PLK2) was significantly increased following treatment with GA101. The increase in the activity of this kinase was specific to GA101 and was unchanged following treatment with Rituximab. Simultaneously there was a significant increase in the phosphorylation of Calumenin at Ser44, a known binding site of PLK2. Samples treated with GA101 for 24 hours exhibited a larger downregulation of pathways associated with calcium signalling compared to Rituximab-treated and control samples.Calumenin, a member of the CREC family, is able to bind 7 Ca2+ ions. It can be localised in the cytosol, in compartments of the secretory pathway, on the plasma membrane or in the extracellular space. We hypothesise that once phosphorylated, this protein bound with Ca2+ is secreted extracellularly, resulting in reduced intracellular Ca2+.In summary we report for the first time that intracellular Ca2+ is significantly decreased as a result of GA101 treatment in cell lines and primary samples. Using a global unbiased approach we have identified a significant increase in PLK2 activity and we observed a large increase in phosphorylation of Calumenin. Our data suggests that these proteins may contribute to the increased direct cell death observed as a result of GA101 treatment. We believe that expression and activity of these proteins are crucial in order to maximise GA101-mediated direct cell death. Ongoing studies are examining the functional roles of PLK2 and Calumenin in the reduction of intracellular Ca2+, comparing Ca2+ levels in response to antibody treatment in primary samples from different CLL subtypes, and assessing and comparing levels of apoptosis following antibody treatment to see whether there is a correlation with reduced intracellular Ca2+. DisclosuresGribben:Celgene: Honoraria; TG Therapeutics: Honoraria; Genentech/Roche: Honoraria; Karyopharm: Honoraria; Kite: Honoraria; Abbvie: Honoraria; Pharmacyclics: Honoraria; Janssen: Honoraria; Acerta: Honoraria.

334-OR: Sex Differences in Protein Phosphorylation in Insulin Resistance in Humans at the Cellular Level

Insulin resistance in muscle precedes and predicts type 2 diabetes in offspring of diabetic parents and is also observed in ~25% of the general population. However, whether these changes represent cell autonomous defects or are secondary to changes in circulating factors is unclear. To define possible cell autonomous determinants of insulin resistance, we have utilized induced pluripotent stem cell (iPSC) differentiated into myoblasts from 20 nondiabetic individuals - 10 insulin sensitive (I-Sen) and 10 insulin resistant (I-Res) as assessed using the steady-state plasma glucose approach. While there was no difference in the ability of the iPSCs to differentiate into myoblasts (iMyos), cells from I-Res individuals show impaired insulin signaling, decreased insulin-stimulated glucose uptake, and glycogen synthase activity compared to iMyos from insulin sensitive individuals, indicating that these cells mirror in vitro the alterations seen in vivo. The global phosphoproteomic analysis using LC-MS/MS uncovered a large network of proteins whose phosphorylation was altered in association with insulin resistance, mostly outside the canonical insulin-signaling cascade. We also observed striking differences in the phosphoproteomic signature of iMyos derived from male versus female subjects. Analysis of the proteins involved reveals that male cells show enhanced phosphorylation of proteins associated with transcription, Rho GTPases, SUMOylation, mRNA splicing, and membrane trafficking, whereas females show a dominance of phosphorylation of proteins involved in the cell cycle, chromatin organization, gene expression, and protein ubiquitination. Taken together, these data demonstrate major alterations in cellular signaling underlying insulin resistance, even in the absence of diabetes, and provide evidence of a super-network of cell signaling differences in males and females that must be considered in understanding cellular regulation in health and disease. Disclosure N. Haider: None. J. Lebastchi: None. T. M. Batista: None. H. Pan: None. J. Dreyfuss: None. I. Carcamo-orive: None. J. Knowles: None. C. Kahn: Advisory Panel; Self; ERX Pharmaceuticals, Kaleido Biosciences, Inc., Consultant; Self; Flagship Pioneering, Sana/Cobalt.

Comprehensive Phosphoproteomic Analysis of Nostoc flagelliforme in Response to Dehydration Provides Insights into Plant ROS Signaling Transduction.

Terrestrial cyanobacteria, originated from aquatic cyanobacteria, exhibit a unique mechanism for drought adaptation during long-term evolution. To elucidate this diverse adaptive mechanism exhibited by terrestrial cyanobacteria from the post-translation modification aspect, we performed a global phosphoproteome analysis on the abundance of phosphoproteins in response to dehydration using Nostoc flagelliforme, a kind of terrestrial cyanobacteria having strong ecological adaptability to xeric environments. A total of 329 phosphopeptides from 271 phosphoproteins with 1168 phosphorylation sites were identified. Among these, 76 differentially expressed phosphorylated proteins (DEPPs) were identified for each dehydration treatment (30, 75, and 100% water loss), compared to control. The identified DEPPs were functionally categorized to be mainly involved in a two-component signaling pathway, photosynthesis, energy and carbohydrate metabolism, and an antioxidant system. We concluded that protein phosphorylation modifications related to the reactive oxygen species (ROS) signaling pathway might play an important role in coordinating enzyme activity involved in the antioxidant system in N. flagelliforme to adapt to dehydration stress. This study provides deep insights into the extensive modification of phosphorylation in terrestrial cyanobacteria using a phosphoproteomic approach, which may help to better understand the role of protein phosphorylation in key cellular mechanisms in terrestrial cyanobacteria in response to dehydration.

SIK2 orchestrates actin-dependent host response upon Salmonella infection

Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.

Insulin Resistance and Gender Define a Cell Autonomous Supernetworkof Protein Phosphorylation

Many hormones and growth factors, including insulin, act through networks of protein phosphorylation. Insulin resistance is an important factor in the pathophysiology of many metabolic disorders. The aim of this study was to uncover the cell autonomous determinants of insulin action and protein phosphorylation using induced pluripotent stem cell (iPSC)-derived myoblasts (iMyos) in vitro. Here, we show that iMyos from non-diabetic individuals in the highest quintile of insulin resistance show impaired insulin signaling, defective insulin-stimulated glucose uptake and decreased glycogen synthase activity compared to iMyos from the insulin sensitive individuals, indicating these cells mirror in vitro the alterations seen in vivo. Global phosphoproteomic analysis uncovered a large network of proteins whose phosphorylation was altered in association with insulin resistance, most outside the canonical insulin-signaling cascade. More surprisingly, we also observed striking differences in the phosphoproteomic signature of iMyos derived from male versus female subjects, involving multiple pathways regulating diverse cellular functions, including DNA and RNA processing, GTPase signaling, and SUMOylation/ubiquitination. These findings provide new insights into the cell autonomous mechanisms underlying insulin resistance in the non-diabetic population and provide evidence of a major, previously unrecognized, supernetwork of cell signaling differences in males and females that must be considered in understanding the molecular basis of sex-based differences in normal physiology and disease.

Phosphoproteome Profiling Revealed the Importance of mTOR Inhibition on CDK1 Activation to Further Regulate Cell Cycle Progression.

The mammalian target of rapamycin (mTOR) functions as a critical regulator of cell cycle progression. However, the underlying mechanism by which mTOR regulates cell cycle progression remains elusive. In this study, we used stable isotope labeling of amino acids in cell culture with a two-step strategy for phosphopeptide enrichment and high-throughput quantitative mass spectrometry to perform a global phosphoproteome analysis of mTOR inhibition by rapamycin. By monitoring the phosphoproteome alterations upon rapamycin treatment, downregulation of mTOR signaling pathway was detected and enriched. Further functional analysis of phosphoproteome revealed the involvement of cell cycle events. Specifically, the elevated profile of cell cycle-related substrates was observed, and the activation of CDK1, MAPK1, and MAPK3 kinases was determined. Second, pathway interrogation using kinase inhibitor treatment confirmed that CDK1 activation operated downstream from mTOR inhibition to further regulate cell cycle progression. Third, we found that the activation of CDK1 following 4-12 h of mTOR inhibition was accompanied by the activation of the Greatwall-endosulfine complex. In conclusion, we presented a high-confidence phosphoproteome map inside the cells upon mTOR inhibition by rapamycin. Our data implied that mTOR inhibition could contribute to CDK1 activation for further regulating cell cycle progression, which was mediated by the Greatwall-endosulfine complex.

Influenza Causes MLKL-Driven Cardiac Proteome Remodeling During Convalescence.

Patients with and without cardiovascular diseases have been shown to be at risk of influenza-mediated cardiac complications. Recent clinical reports support the notion of a direct link between laboratory-confirmed influenza virus infections and adverse cardiac events. Define the molecular mechanisms underlying influenza virus-induced cardiac pathogenesis after resolution of pulmonary infection and the role of necroptosis in this process. Hearts from wild-type and necroptosis-deficient (MLKL [mixed lineage kinase domain-like protein]-KO) mice were dissected 12 days after initial influenza A virus (IAV) infection when viral titers were undetectable in the lungs. Immunofluorescence microscopy and plaque assays showed presence of viable IAV particles in the myocardium without generation of interferon responses. Global proteome and phosphoproteome analyses using high-resolution accurate mass-based LC-MS/MS and label-free quantitation showed that the global proteome as well as the phosphoproteome profiles were significantly altered in IAV-infected mouse hearts in a strain-independent manner. Necroptosis-deficient mice had increased survival and reduced weight loss post-IAV infection, as well as increased antioxidant and mitochondrial function, indicating partial protection to IAV infection. These findings were confirmed in vitro by pretreatment of human and rat myocytes with antioxidants or necroptosis inhibitors, which blunted oxidative stress and mitochondrial damage after IAV infection. This study provides the first evidence that the cardiac proteome and phosphoproteome are significantly altered post-pulmonary influenza infection. Moreover, viral particles can persist in the heart after lung clearance, altering mitochondrial function and promoting cell death without active replication and interferon responses. Finally, our findings show inhibition of necroptosis or prevention of mitochondrial damage as possible therapeutic interventions to reduce cardiac damage during influenza infections. Graphic Abstract: A graphic abstract is available for this article.

Rapid Shotgun Phosphoproteomics Analysis.

In this chapter, we describe a rapid workflow for the shotgun global phosphoproteomics analysis. The strategy is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field by high-intensity focused ultrasound (HIFU) coupled to titanium dioxide (TiO2) selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX), and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a high-resolution mass spectrometer (LTQ-Orbitrap XL). The strategy was optimized for the global phosphoproteome analysis of Jurkat T-cells. Using this accelerated workflow, HIFU-TiO2-SCX-LC-MS/MS, 15,367 phosphorylation sites from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins can be efficiently identified in less than 15h.

Proteome and phosphoproteome characterization of liver in the postprandial state from diet-induced obese and lean mice

A metabolic consequence of obesity is hepatosteatosis, which can develop into more serious diseases in the non-alcoholic fatty liver disease (NAFLD) spectrum. The goal of this study was to identify the protein signature of liver in the postprandial state in obesity compared to leanness. The postprandial state is of interest due to the central role of the liver in regulating macronutrient and energy homeostasis during the fed-fast cycle and lack of previously reported controlled studies in the postprandial state. Therefore, we assessed the proteome and phosphoproteome of liver in the postprandial state from diet-induced obese (DIO) and lean mice using untargeted LC-MS/MS analysis. We identified significant alterations in the levels of proteins involved in fatty acid oxidation, activation, and transport, as well as proteins involved in energy metabolism including ketogenesis, tricarboxylic acid cycle, and electron transport chain in liver of DIO compared to lean mice. Additionally, phosphorylated proteins in liver of DIO and lean mice reflect possible regulatory mechanisms controlling fatty acid metabolism and gene expression that may contribute to hepatic metabolic alterations in obesity. Our data indicates PPARα-mediated transcriptional regulation of lipid metabolism and adaptation to hepatic lipid overload. The results of this study expand our knowledge of the molecular changes that occur in liver in the postprandial state in obesity compared to leanness. SignificanceProteome and phosphoproteome studies of liver in a controlled postprandial state in obesity and leanness are lacking; however, this information is crucial to understanding how obesity-associated hepatosteatosis influences postprandial nutrient and energy metabolism. In this global shotgun proteome and phosphoproteome analysis, we identified unique protein signatures defining obesity and leanness in liver in the postprandial state and identified potential mechanisms contributing to hepatic metabolic alterations in obesity. The results of this study provide a foundation to focus future experiments on the contribution of altered protein and phosphorylation patterns to postprandial metabolism in obesity-associated hepatosteatosis.

Abstract 15386: Integrated Phosphoproteome and Transcriptome Analysis of Biased Agonists at CXCR3 Reveals Differential Cellular Outputs of Inflammation

Background and Objective: G-protein-coupled receptors (GPCRs) are the most common cell surface receptor class and are known to influence nearly every biological process. GPCRs signal through downstream effectors such as G proteins and β-arrestins. Certain ligands drive activation of G protein over β-arrestin signaling, or vice versa - a phenomenon known as biased agonism. To investigate the mechanisms and implications of biased agonism, we studied CXCR3, a chemokine receptor expressed on T cells, which binds three biased endogenous ligands, CXCL9, CXCL10, and CXCL11. Importantly, CXCR3 has been implicated in atherosclerosis and other inflammatory diseases. Methods: To obtain a broad view of biased signaling at CXCR3, we performed global phosphoproteomic and transcriptomic analyses of CXCR3-transfected HEK 293 cells and activated human CD8+ T-cells, respectively, treated with CXCL9, CXCL10, and CXCL11. To assess the phosphoproteome, we utilized an antibody-free mass spectrometry approach with high-pressure, high-resolution separation and intelligent selection. RNA-seq was performed to assess transcriptomic changes in activated CD8+ T-cells. Results: Our analyses revealed 1532 phosphosites and 887 transcripts differentially regulated between treatment groups. Gene Set Enrichment Analysis (GSEA) of CXCL11-treated cells revealed enrichment of pro-inflammatory targets including the Myc and TNF-α pathways via NFKB not observed in CXCL10- or CXCL9-treated cells. GSEA of CXCL10- and CXCL11-treated cells also revealed enrichment of the IL6-JAK-STAT3 pathway, linked to chronic inflammatory conditions. Kinase-Substrate Enrichment Analysis (KSEA) revealed differential phosphorylation activity associated with multiple kinases including AURKB (CXCL9), ROCK1 (CXCL9, 10, 11), CDK9 (CXCL10), and ATM (CXCL10, 11) - all known to regulate pro-inflammatory targets. Several phosphoproteins were transcription factors, including p53, known to mediate IL-6 production and atherosclerosis pathogenesis. Conclusion: Together, our integrated analysis of phosproteomic and transcriptomic data reveal the extent of differential cellular outputs produced by biased agonists at CXCR3 in modulating targets implicated in inflammatory diseases.