Plants are sessile organisms and constantly encounter a myriad of pathogens; therefore, they rely on highly effective defense system for their survival. Our understanding of how plant immunity is triggered and regulated has seen tremendous progress over the last two decades, with many important players identified in the model systems, Arabidopsis thaliana. Mitogen activated protein kinases play a central role in signal transduction in biotic and abiotic stresses. MAPK pathways are regulated by three-interlinked protein kinases (MAPKKK, MAPKK, MAPK), which are sequentially activated by phosphorylation. The activation of the three MAPKs MPK3, MPK4 and MPK6 is one of the earliest cellular responses following pathogen attack leading to the phosphorylation of appropriate cytosolic or nuclear targets to regulate cellular processes. However, only few targets of MPK3, MPK4 and MPK6 have been identified and validated so far and many MAPK substrates remain to be discovered. We performed largescale phosphoproteomics on mock treated and flg22 treated WT and the three loss-of-function mutants mpk3, mpk4 and mpk6 to identify novel MAPKs substrates and their cellular functions in response to pathogen attack. We identify and validated some of the differentially phosphorylated cytosolic and chromatin targets of MPK3, MPK4 and MPK6. DEK2, a nuclear protein involved in multiple chromatin-related processes, was identified in the phosphoproteomics screen as an in vivo target of MPK6 and it interacts in planta and is phosphorylated in vitro by the three immune MAPKs. dek2 loss-of-function mutants were susceptible to bacterial as well as fungal pathogens. Additionally, transcriptome data of the dek2-1 mutant show that DEK2 is a transcriptional repressor inclusive of defense related genes and hormone synthesis and signaling genes. We determined that DEK2 is a reader of the histone mark, H3K9me1, by Microscale thermophoresis. From ChIP-Seq analysis, DEK2 was found to be enriched at class I TCP binding motif regions. We further need to determine whether DEK2 binds to TCP transcription factors directly or indirectly. Finally, based on our data we postulate a hypothetical working model for the function of DEK2 as a transcriptional repressor and a reader of H3K9me1 mark.

No description available.

Malaria is a life-threatening disease caused by Plasmodium parasites that are spread through the bites of infected mosquito vectors. It is a worldwide pandemic that threatens 3.4 billion people annually. Currently, there are only a few validated Plasmodium drug targets, while drug resistance continues to rise. This marks the urgency for the development of novel parasite-specific therapeutics. Plasmodium falciparum diverges from the paradigm of the eukaryotic cell cycle by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for the design of rational intervention strategies. To achieve this goal, we performed an isobaric tag-based approach for a system-wide quantitative analysis of protein expression and site-specific phosphorylation events of the Plasmodium asexual developmental cycle in the red blood cells. This study identified 2,767 proteins, 1,337 phosphoproteins, and 6,293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Because the links between Plasmodium protein kinases as key cell cycle regulators to cellular events are largely unknown, it is of importance to define their cognate physiological substrates. To test the hypothesis that genetic screening would be a useful approach for discovery of candidate substrates of a protein kinase, we used the orphan kinase PfPK7 as a model. Our comparison of the phosphoproteome profiles between the wild-type 3D7 and PfPK7- parasites identified 146 proteins with 239 phosphorylation sites exhibiting decreased phosphorylation in the absence of PfPK7 at the developmental stages where nuclear division and merozoite formation occur. Further analysis of the decreased phosphorylated events revealed three motifs that are enriched among phosphorylated sites in proteins that are down regulated. In vitro kinase assays were done to validate the potential substrates of PfPK7 and to elucidate the signaling events that are regulated by PfPK7. In parallel to our experimental analysis, we used a computational approach for substrate prediction from our phosphoproteome dataset. This analysis identified 43 distinct phosphorylation motifs and a range of proline-directed potential MAPK/CDK substrates. To identify substrates/ interactors of Plasmodium CDK-like kinases, we also used HA-tagged CDK-like kinases, PfPK6 and Pfmrk lines. Co-immunoprecipitation of the HA-tagged PfPK6 and Pfmrk baits, followed by mass spectrometric analyses, identified the components of the protein interaction complexes of these kinases. Our analyses of HA-PfPK6 and HA-Pfmrk immunoprecipitates identified 15 and 21 proteins in the interaction complex, respectively. The ability of recombinant PfPK6 and Pfmrk to interact and/or utilize any of the proteins identified in the interaction complex as substrates was verified through in vitro kinase assays and pull-down analysis. This study is the most comprehensive definition of the constitutive and regulated expression of the Plasmodium proteome during the intraerythrocytic developmental cycle, and offered an insight into the dynamics of phosphorylation during the asexual cycle progression [1]. In summary, this study has 1) defined the constitutive and regulated expression of the Plasmodium proteome during its asexual life cycle, 2) demonstrated that fluctuation and reversible phosphorylation is important for the regulation of P. falciparum*s unique cell cycle, 3) provided the foundation for quantitative phosphoproteomic analysis of kinase negative mutants to understand their function, 4) provided a major step towards defining kinase-substrate pairs operative within parasite*s signaling networks, and 5) generated a preliminary interactome for PfPK6.

E-selectin is a transmembrane endothelium adhesion protein involved in rolling, arrest and migration of leukocytes as well as in the metastasis of many cancer types. Previous reports suggested that the interactions between E-selectin and its ligands transduce signals into migrating leukocytes and in E-selectin expressing endothelial cells. This study investigates the signaling pathways involved in E-selectin binding to ligands on leukocytes. Using recombinant soluble E-selectin constructs, we simulated the binding of E-selectin to its ligand(s) to reveal important signaling pathways triggered upon these interactions in acute myeloid leukemia (AML) cells. Since phosphorylation is the major post-translational modification, we examined the changes in the phosphorylation profile in tyrosine residues. We found a time-dependent reduction in the phosphotyrosine levels upon E-selectin binding to the AML cell line, KG-1a. The results of this study revealed two tyrosine phosphatases with altered activity after E-selectin treatment. The first is a cytoplasmic, dual-specific, phosphatase known as PTEN which is involved in controlling cell survival and proliferation. The second is CD45, which is a major component of the leukocytes cell membrane responsible for antigen receptor signaling. A more global phosphoproteomics analysis in AML cells revealed large scale changes in the phosphorylation levels after E-selectin treatment. In particular, 2259 phosphorylated proteins were identified, 530 of which portray significant changes in their phosphorylation status. The majority of those proteins are related to nuclear functions and are involved in pathways crucial for the cell cycle. Knowing that E-selectin binding stimulates chemoresistance in cancer cells, the findings of this project can contribute to the identification of multiple pathways responsible for this phenomenon and help towards the development of drugs that may inhibit such pathways in controlling disease.

Medical diagnosis is the process of attempting to determine and/or identify a possible disease or disorder. This process is revealed by biomarkers, defined by The Food and Drug Administration (FDA) as “characteristics that are objectively measured and evaluated as indicators of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”. The process of biomarker discovery has been boosted in the last years by proteomics, a research discipline that takes a snapshot of the entire wealth of proteins in an organism/ tissue/ cell/ body fluid. An implementation of the analysis methods can help in isolate proteins present in the low range of concentrations, such as biomarkers very often are. An established biomarker can further be measured with the help of biosensors, devices that can be employed in the point-of care diagnostics. This PhD thesis shows and discusses the results of three projects in the field of protein biomarkers discovery and quantification. The first project exploited proteomics techniques to find relevant protein markers for Intrauterine Growth Restriction (IUGR) in cordonal blood serum (UCS) and amniotic fluid (AF). A 14 proteins in UCS and 11 in AF were successfully identified and found to be differentially expressed. Molecularly Imprinted Polymers (MIPs) directed towards proteins and peptides containing phosphotyrosine were then produced, with the final goal of selectively extracting phosphopeptides from a peptide mixture. An alteration of the phosphorylation pattern is in fact often associated to important diseases such as cancer. The polymers were produced as nanoparticles, that were characterised with Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). A recipe was also tested for binding capacity towards phosphotyrosine. A Surface Plasmon Resonance (SPR) biosensor to quantify hepcidin hormone was finally produced. This is the major subject in iron homeostasis in vertebrates and marker of iron unbalance diseases. A calibration curve was made and affinity/kinetic parameters for the ligand employed were measured.

Glucocorticoids (GCs) are a class of steroid hormone that play essential roles in development, glucose homeostasis, and reducing inflammation. Clinically, GCs are potent anti-inflammatory and immunosuppressive agents used to treat a variety of diseases. However, the therapeutic benefit of GCs is negatively impacted by the induction of severe side effects. In this thesis, I present two studies that have contributed to the understanding of the non-genomic actions of GCs. GCs inhibit cell migration by a non-transcriptional pathway involving HDAC6: A negative side effect of GC therapy is impaired wound healing which is ascribed to inhibited cell migration. Using live-cell microscopy, I show that GCs inhibit cell migration within 30 minutes of administration. GCs alter the dynamics of the microtubule network through rapid induction of tubulin acetylation (by inhibition of HDAC6) which increases microtubule stability and slows cell movement. The inhibitory effect of GCs on cell migration is reversed by overexpressing HDAC6. Using quantitative imaging, I identified a rapid ligand-dependent association of the GR and HDAC6 within the cytoplasm that is absent in the nucleus. However, a very small proportion of HDAC6 enters the nucleus post-GC treatment, suggesting that HDAC6 accompanies the GR during nuclear translocation. This study demonstrates that GCs rapidly inhibit cell migration by a non-transcriptional mechanism involving HDAC6. Investigating the rapid effects of GCs on the phosphoproteome: Non-steroidal GCs are useful tool compounds to dissect glucocorticoid receptor (GR) activity. Here, I investigated the early, rapid effect of GCs on the phosphoproteome of A549 cells using SILAC-based phosphoproteomics. A consistent spectrum of phosphoproteins was differentially regulated by GC within 10 minutes of administration, notably including regulators of RNA polymerase II, chromatin remodifying proteins, transcription factors, cytoskeletal modifiers, regulators of intracellular calcium signalling and endocytosis. These phosphoproteins were validated by western blotting. This study shows a clear early effect of GCs on the phosphoproteome with implications for non-specific, non-transcriptional activity of GCs.

Alzheimer’s disease (AD) is the most prevalent form of dementia affecting the elderly, and as the aging population increases the social and economic burden of AD grows substantially. Pathological hallmarks of AD include the accumulation of extracellular amyloid plaques and intracellular neurofibrillary tangles (NFTs), as well as significant neuron loss. Amyloid plaques consist of aggregated amyloid beta (Aβ) peptide, which is generated from the proteolytic processing of amyloid precursor protein (APP) in addition to several other peptides. While the processing of APP has been characterized, its primary physiological function and its involvement in AD pathology are poorly understood. Developing a greater understanding of the function of APP, and the molecular and cellular functions it is involved in or other proteins it is associated with, could provide insight into its role in AD pathology. To investigate the function of APP695, the neuronal isoform of APP, we used mass spectrometry to compare changes in protein expression and phosphorylation between APP-null B103 and APP695-expressing B103-695 rat neuroblastoma cells. Mass spectrometry-based proteomics has become a powerful technique for the unbiased identification of proteins from complex mixtures. Quantitative proteomics using labeling techniques, such as stable isotope labeling by amino acids in cell culture (SILAC), allow relative quantitation of multiple samples at once. More recently, with advances in mass spectrometer technology, label-free quantitation has become a reliable quantitative proteomics approach. Additionally, mass spectrometry can be used for the analysis of post-translational modifications, such as phosphorylation, a dynamic modification involved in the regulation of many cellular processes. Phosphoproteomics identifies site-specific phosphorylation and surrounding sequence information, which can be used for consensus motif analysis to provide further information about potential changes in kinase activity. Identifying changes in phosphorylation and kinase activity also provides information about signaling pathways and functions that may be affected by APP695 expression. Comprehensive proteomic and phosphoproteomic datasets can be used to gain insight into the molecular mechanisms that may be regulated by APP695 expression, or involved in AD progression and pathology, leading to the development of novel therapeutic and preventative strategies for AD. Proteomic and phosphoproteomic analysis of B103 and B103-695 cells identified several significant protein expression and phosphorylation changes that may be mediated by APP695-expression. Global-scale proteomic analysis identified increased expression of Ras and ƴ-synuclein in B103-695 cells, which was further validated in human AD brain tissue. Phosphoproteomic analysis showed increased phosphorylation of Histone H4 at Ser47, and led to the investigation of PCTAIRE-2 (Cdk17), and PCTAIRE-3 (Cdk18) expression, which were all shown to be increased in AD transgenic mouse tissue, culture primary rat neurons treated with Aβ, as well as mild cognitive impairment (MCI) and AD human brain tissue. Label-free quantitative proteomics was used for the analysis of human brain tissue from the cortex of individuals affected by AD, MCI, Parkinson’s disease (PD), and progressive supranuclear palsy (PSP) compared to cognitively normal, control samples. A number of differentially expressed proteins were identified in AD, MCI, PD, and PSP tissue. Bioinformatic analysis of the comprehensive proteomic datasets from AD, MCI, PD, and PSP human brain tissue identified several proteins consistent with corresponding disease pathology and neurodegeneration, such as inflammatory proteins. While some of the molecular and cellular functions were unique among neurodegenerative diseases, there also appears to be overlap of affected functions, suggesting there may be a more common mechanism of neurodegeneration.

PANcreatic DERived factor (PANDER, FAM3B) is a member of a superfamily of FAM3 proteins that are uniquely structured and strongly expressed from the endocrine pancreas and co-secreted with insulin. Unique animal models available to our lab have indicated that PANDER can induce a selective hepatic insulin resistant (SHIR) phenotype whereby insulin signaling is blunted yet lipogenesis is increased. The complexity of the biological networks involved with this process warranted the logical approach of employing quantitative mass spectrometry based proteomic analysis using stable isotope labeling of amino acids in cell culture (SILAC) to identify the global proteome differences between the PANDER transgenic (TG) overexpressing murine model to matched wild-type mice under three metabolic states (fasted, fed and insulin stimulated). Additionally, this technique was used to compare the hepatic proteome of mice on a high fat diet to elucidate early and late mechanisms of disease progression. The “spike-in” process was employed by equal addition of lysate obtained from livers of heavy L-Lysine (13C6, 97%) fed mice to the mice liver protein lysate (PANTG and WT) for relative quantitative analysis. Upon acquisition of the dataset by use of liquid chromatography tandem mass spectrometry (LC-MS/MS, LTQ Orbitrap), geometric means and Uniprot Protein identification numbers were uploaded to Ingenuity Pathway Analysis (IPA) to reveal the effect of PANDER on hepatic signaling. IPA identified lipid metabolism and fatty acid synthesis as top cellular functions differentially altered in all metabolic states. Several molecules with a role in lipid metabolism were identified and include FASN, ApoA1, ApoA4, SCD1, CD36, CYP7A1 and ACC. Furthermore, central to the differentially expressed proteins was the revealed activation of the liver X receptor (LXR) pathway. In summary, our SILAC proteomic approach has elucidated numerous previously unidentified PANDER induced molecules and pathways resulting in increased hepatic lipogenesis. In addition, we have demonstrated strong utility of this approach in comprehensively phenotyping animal models of hepatic insulin resistance. PANDER may potentially propagate pro-hepatic lipogenic effects by LXR activation in contrast to increased LXRα expression. This can be evaluated through the use of LXR agonists (T0901317) antagonists (GSK 2033). LXR activity can be measured by luciferase assays using an LXRE response plasmid. Our central hypothesis is that PANDER induces activation of LXR and is measured and predicted in our line of experiments. In general, PANDER induced LXR activation will be enhanced by T0901317 and diminish effects of GSK 2033 along with direct correlation of downstream metabolic effects such as increased hepatic lipogenesis and fatty acid metabolism. Taken together, PANDER strongly impacts hepatic lipid metabolism and may induce a SHIR phenotype via the LXR pathway. Additionally, phosphoproteomic analysis uncovered large-scale differences in protein phosphorylation states as PANDER impacts insulin signaling. A notable finding was the increased phosphorylation of glycogen synthase (GSK), possibly responsible for the decreased hepatic glycogen content in the PANTG mouse. In an effort to map out critical molecules involved in non-alcoholic fatty liver disease (NAFLD) pathogenesis, the same proteomic approach was carried out, providing a unique dataset of differentially expressed hepatic proteins due to a high at diet.

<p>Protein tyrosine phosphorylation is an essential posttranslational modification that controls cell signaling involving various biological processes, including cell growth, proliferation, migration, survival, and death. Balancing tyrosine phosphorylation levels is necessary for normal and pathological states, and this reversible mechanism occurs through protein tyrosine kinases and phosphatases. Advancements in instrumentation and applying conventional biochemical and genetic methods have led to cell signaling studies and pharmaceutical development discoveries. However, there is still a lack of understanding of tyrosine phosphatases' mechanisms, substrates, and activities within complex networks. The challenges remain in the tyrosine phosphatase field due to the low abundance and dynamic nature, sample preparation steps, and sensitivity to detect tyrosine phosphorylation events. Although mass spectrometry (MS)-based phosphoproteomics has allowed the identification of thousands of phosphotyrosine sites in a single run, protein phosphorylation poses another analysis caveat of dissecting complex phosphorylation signaling pathways involved in healthy cellular processes similarly in disease pathogenesis. This dissertation discusses strategies for improving tyrosine phosphatase sample preparation and identifying the tyrosine phosphatases' direct substrates. Chapter one is an overview of current techniques to study tyrosine phosphatases. In contrast, chapters two and three highlight the work that has been done to identify the direct substrates of phosphatase SHP2 and PTP1B, respectively, whose dysregulation leads to the development of cancers.</p> <p>In chapter 2, we describe a novel method that incorporated three separate MS-based experiments to identify the direct substrates of phosphatase SHP2: immunoprecipitation of substrate trapping mutants complex, <i>in vivo </i>global phosphoproteomics, and <i>in vitro</i> dephosphorylation of SHP2 phosphatase substrates. With immunoprecipitation of substrate trapping mutant experiment, weak and transient phosphatase-substrate interactions were detected by mass spectrometry after being stabilized by substrate trapping mutant method. This experiment not only identified the interactions between phosphatase and substrates but also revealed phosphotyrosine sites that are potentially protected in the substrate trapping mutant. We identified 80 phosphotyrosine proteins that showed upregulated in SHP2 mutant samples, and GAB1, GAB2, IRS1, SIRPA, and MPZL1 were examined in our list, which are reported SHP2 substrates. In the second experiment in parallel, we explored the global phosphorylation in HEK293 cells stimulated by epidermal growth factor. Peptides containing phosphotyrosine residues were captured by immobilized anti-pY PT-66 antibody and analyzed by LC-MS/MS. The results provided information on how SHP2 regulates downstream protein tyrosine phosphorylation and global phosphotyrosine response initiated by EGF. We used SHP2 substrate trapping mutant to isolate phosphotyrosine-containing proteins to serve as a SHP2 substrate pool for an <i>in vitro </i>phosphatase assay, then analyzed by LC-MS/MS. Finally, the overlap of the three separate MS-based experiments gave us the final list of high-confidence SHP2 substrates. DOK1 was validated to be a direct SHP2 substrate. </p> Chapter 3 describes a novel method that integrates <i>in vivo </i>global phosphoproteomics perturbed by PTP1B inhibitor and stimulated by insulin with <i>in vitro </i>kinetic profile of PTP1B phosphatase to identify its substrates. We were able to identify 114 phosphotyrosine proteins that showed upregulated in PTP1B inhibitor and insulin-treated sample in <i>in vivo </i>global phosphoproteomics experiment. CTTN, EGFR, FER, IRS1, PTPN11, SRC, TYK2, PKM, GAB1, GAB2, and INSR were examined, which are PTP1B reported substrates. <a>In <i>in vitro </i>kinetic profile of the PTP1B phosphatase experiment, we utilized dimethyl labeling to quantify the PTP1B dephosphorylation rate. No PTP1B substrate motif consensus was observed in the labeling experiments. We finally overlapped <i>in vivo </i>and <i>in vitro </i>experiments to identify PTP1B <i>bona fide </i>substrates with high confidence.</a>

Crohn’s disease (CD), a chronic inflammatory condition of the gut affecting 1:1000 of western populations, is thought to arise from a dysregulated immune response in a genetically susceptible individual. Polymorphisms in the ligand recognition domain of an intracellular pattern recognition receptor (PRR), NOD2, remain the strongest genetic risk factor for the development of CD. NOD2 directs autophagy in human DCs to facilitate bacterial destruction and antigen presentation; the CD-variant-NOD2 shows defects in this pathway. Recent work in the laboratory has demonstrated NOD2 signals to control expression induction of microRNA-29, which is impaired in cells from CD patients expressing CD NOD2-variants, and among other immunoregulatory effects, microRNA-29 suppresses IL-12p40/IL-23. Thus NOD2 directs key anti-microbe and immunoregulatory functions whose breakdown in the presence of CD-variant-NOD2 could act as a trigger for inflammation in this disease. In comparison with other PRRs, relatively little is understood of the hardwiring of NOD2 signalling, the mechanism of NOD2-mediated autophagy induction, the means by which NOD2 recruits a signalling platform within the cytosol and the mechanism of synergy with other PRRs and the inflammasome. In this work I used quantitative proteomics to map the NOD2 signalling cascade and its cross-talk with TLR2, demonstrating novel mediators: LCP1, a plastin, reduced phagocytosis of bacteria but did not alter bacterial killing, and aided control of the release of MCP1 and may be involved in IL-12p40 release. SHP1, a phospho-tyrosine phosphatase, is required for the propagation of signalling cascades via p38, p44/42 MAPK and NF-κB. It controls release of MIP1β and IL-12p40. HMGB1, an alarmin, is dephosphorylated on NOD2 stimulation and would result in changes to cellular location of HMGB1. Lastly, DAPK1, a serine/threonine kinase, is associated with HLA-DM loading compartment on NOD2 triggering, but does not alter CLIP levels on the surface of the cells. Thus, defining the hard wiring of NOD2 signalling in healthy donors, in comparison with CD donors expressing variant NOD2, is important to define targets amenable to drug design within this pathway.