Kizhake V. Soman

Src activation by adrenoreceptors is a key switch for tumour metastasis

Norepinephrine (NE) can modulate multiple cellular functions important for cancer progression; however, how this single extracellular signal regulates such a broad array of cellular processes is unknown. Here, we identify Src as a key regulator of phosphoproteomic signaling networks activated in response to beta-adrenergic signaling in cancer cells. These results also identify a new mechanism of Src phosphorylation that mediates beta-adrenergic/PKA regulation of downstream networks, thereby enhancing tumor cell migration, invasion and growth. In human ovarian cancer samples, high tumoral NE levels were correlated with high pSrcY419 levels. Moreover, among cancer patients, the use of beta blockers was significantly associated with reduced cancer-related mortality. Collectively, these data provide a pivotal molecular target for disrupting neural signaling in the tumor microenvironment.

Homology modeling and characterization of IgE binding epitopes of mountain cedar allergen Jun a 3.

The Jun a 3 protein from mountain cedar (Juniperus ashei) pollen, a member of group 5 of the family of plant pathogenesis-related proteins (PR-proteins), reacts with serum IgE from patients with cedar hypersensitivity. We used the crystal structures of two other proteins of this group, thaumatin and an antifungal protein from tobacco, both approximately 50% identical in sequence to Jun a 3, as templates to build homology models for the allergen. The in-house programs EXDIS and FANTOM were used to extract distance and dihedral angle constraints from the Protein Data Bank files and determine energy-minimized structures. The mean backbone deviations for the energy-refined model structures from either of the templates is <1 A, their conformational energies are low, and their stereochemical properties (determined with PROCHECK) are acceptable. The circular dichroism spectrum of Jun a 3 is consistent with the postulated beta-sheet core. Tryptic fragments of Jun a 3 that reacted with IgE from allergic patients all mapped to one helical/loop surface of the models. The Jun a 3 models have features common to aerosol allergens from completely different protein families, suggesting that tertiary structural elements may mediate the triggering of an allergic response.

Quantification of cysteinyl S-nitrosylation by fluorescence in unbiased proteomic studies

Cysteinyl S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins because of physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that uses accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo) and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia/reperfusion model and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the techniques power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses.

Role of Peroxiredoxin 1 and Peroxiredoxin 4 in Protection of Respiratory Syncytial Virus-Induced Cysteinyl Oxidation of Nuclear Cytoskeletal Proteins

ABSTRACT The respiratory epithelium plays a central role in innate immunity by secreting networks of inflammatory mediators in response to respiratory syncytial virus (RSV) infection. Previous proteomic studies focusing on the host cellular response to RSV indicated the existence of a nuclear heat shock response and cytoplasmic depletion of antioxidant proteins in model type II-like airway epithelial cells. Here, we increased the depth of nuclear proteomic interrogation by using fluorescence difference labeling followed by liquid isoelectric focusing prefractionation/two-dimensional gel electrophoresis (2-DE) to identify an additional 41 proteins affected by RSV infection. Surprisingly, we found inducible oligomers and shifts in isoelectric points for peroxiredoxin 1 (Prdx-1), Prdx-3, and Prdx-4 isoforms without changes in their total abundance, indicating that Prdxs were being oxidized in response to RSV. To address the role of Prdx-1 and Prdx-4 in RSV infection, isoforms were selectively knocked down by small interfering RNA (siRNA) transfection. Cells lacking Prdx-1, Prdx-4, or both showed increased levels of reactive oxygen species formation and a higher level of protein carbonylation in response to RSV infection. Using a novel saturation fluorescence labeling 2-DE analysis, we showed that 15 unique proteins had enhanced oxidative modifications of at least >1.2-fold in the Prdx knockdowns in response to RSV, including annexin A2 and desmoplakin. Our results suggest that Prdx-1 and Prdx-4 are essential for preventing RSV-induced oxidative damage in a subset of nuclear intermediate filament and actin binding proteins in epithelial cells.

Discovery Proteomics and Nonparametric Modeling Pipeline in the Development of a Candidate Biomarker Panel for Dengue Hemorrhagic Fever

Secondary dengue viral infection can produce capillary leakage associated with increased mortality known as dengue hemorrhagic fever (DHF). Because the mortality of DHF can be reduced by early detection and intensive support, improved methods for its detection are needed. We applied multidimensional protein profiling to predict outcomes in a prospective dengue surveillance study in South America. Plasma samples taken from initial clinical presentation of acute dengue infection were subjected to proteomics analyses using ELISA and a recently developed biofluid analysis platform. Demographics, clinical laboratory measurements, nine cytokines, and 419 plasma proteins collected at the time of initial presentation were compared between the DF and DHF outcomes. Here, the subjects gender, clinical parameters, two cytokines, and 42 proteins discriminated between the outcomes. These factors were reduced by multivariate adaptive regression splines (MARS) that a highly accurate classification model based on eight discriminant features with an area under the receiver operator curve (AUC) of 0.999. Model analysis indicated that the feature–outcome relationship were nonlinear. Although this DHF risk model will need validation in a larger cohort, we conclude that approaches to develop predictive biomarker models for disease outcome will need to incorporate nonparametric modeling approaches. Clin Trans Sci 2012; Volume #: 1–13

Toward the proteome of the human peripheral blood eosinophil

Eosinophils (EOSs) are granular leukocytes that have significant roles in many inflammatory and immunoregulatory responses, especially asthma and allergic diseases. We have undertaken a fairly comprehensive proteomic analysis of purified peripheral blood EOSs from normal human donors primarily employing 2‐DE with protein spot identification by MALDI‐MS. Protein subfractionation methods employed included IEF (Zoom® Fractionator) and subcellular fractionation using differential protein solubilization. We have identified 3141 proteins, which had Mascot expectation scores of 10−3 or less. Of these 426 were unique and non‐redundant of which 231 were novel proteins not previously reported to occur in EOSs. Ingenuity Pathway Analysis showed that some 70% of the non‐redundant proteins could be subdivided into categories that are clearly related to currently known EOS biological activities. Cytoskeletal and associated proteins predominated among the proteins identified. Extensive protein posttranslational modifications were evident, many of which have not been previously reported that reflected the dynamic character of the EOS. This data set of eosinophilic proteins will prove valuable in comparative studies of disease versus normal states and for studies of gender differences and polymorphic variation among individuals.

Liver proteomics in progressive alcoholic steatosis

Fatty liver is an early stage of alcoholic and nonalcoholic liver disease (ALD and NALD) that progresses to steatohepatitis and other irreversible conditions. In this study, we identified proteins that were differentially expressed in the livers of rats fed 5% ethanol in a Lieber-DeCarli diet daily for 1 and 3 months by discovery proteomics (two-dimensional gel electrophoresis and mass spectrometry) and non-parametric modeling (Multivariate Adaptive Regression Splines). Hepatic fatty infiltration was significantly higher in ethanol-fed animals as compared to controls, and more pronounced at 3 months of ethanol feeding. Discovery proteomics identified changes in the expression of proteins involved in alcohol, lipid, and amino acid metabolism after ethanol feeding. At 1 and 3 months, 12 and 15 different proteins were differentially expressed. Of the identified proteins, down regulation of alcohol dehydrogenase (-1.6) at 1 month and up regulation of aldehyde dehydrogenase (2.1) at 3 months could be a protective/adaptive mechanism against ethanol toxicity. In addition, betaine-homocysteine S-methyltransferase 2 a protein responsible for methionine metabolism and previously implicated in fatty liver development was significantly up regulated (1.4) at ethanol-induced fatty liver stage (1 month) while peroxiredoxin-1 was down regulated (-1.5) at late fatty liver stage (3 months). Nonparametric analysis of the protein spots yielded fewer proteins and narrowed the list of possible markers and identified d-dopachrome tautomerase (-1.7, at 3 months) as a possible marker for ethanol-induced early steatohepatitis. The observed differential regulation of proteins have potential to serve as biomarker signature for the detection of steatosis and its progression to steatohepatitis once validated in plasma/serum.

Arsenal of elevated defense proteins fails to protect tomato against Verticillium dahliae

Although the hypersensitive reaction in foliar plant diseases has been extensively described, little is clear regarding plant defense strategies in vascular wilt diseases affecting numerous economically important crops and trees. We have examined global genetic responses to Verticillium wilt in tomato (Lycopersicon esculentum Mill.) plants differing in Ve1 resistance alleles. Unexpectedly, mRNA analyses in the susceptible plant (Ve1−) based on the microarrays revealed a very heroic but unsuccessful systemic response involving many known plant defense genes. In contrast, the response is surprisingly low in plants expressing the Ve1+ R-gene and successfully resisting the pathogen. Similarly, whole-cell protein analyses, based on 2D gel electrophoresis and mass spectrometry, demonstrate large systemic increases in a variety of known plant defense proteins in the stems of susceptible plants but only modest changes in the resistant plant. Taken together, the results indicate that the large systemic increases in plant defense proteins do not protect the susceptible plant. Indeed, since a number of the highly elevated proteins are known to participate in the plant hypersensitive response as well as natural senescence, the results suggest that some or all of the disease symptoms, including ultimate plant death, actually may be the result of this exaggerated plant response.

Improved Detection of Invasive Pulmonary Aspergillosis Arising during Leukemia Treatment Using a Panel of Host Response Proteins and Fungal Antigens.

Invasive pulmonary aspergillosis (IPA) is an opportunistic fungal infection in patients undergoing chemotherapy for hematological malignancy, hematopoietic stem cell transplant, or other forms of immunosuppression. In this group, Aspergillus infections account for the majority of deaths due to mold pathogens. Although early detection is associated with improved outcomes, current diagnostic regimens lack sensitivity and specificity. Patients undergoing chemotherapy, stem cell transplantation and lung transplantation were enrolled in a multi-site prospective observational trial. Proven and probable IPA cases and matched controls were subjected to discovery proteomics analyses using a biofluid analysis platform, fractionating plasma into reproducible protein and peptide pools. From 556 spots identified by 2D gel electrophoresis, 66 differentially expressed post-translationally modified plasma proteins were identified in the leukemic subgroup only. This protein group was rich in complement components, acute-phase reactants and coagulation factors. Low molecular weight peptides corresponding to abundant plasma proteins were identified. A candidate marker panel of host response (9 plasma proteins, 4 peptides), fungal polysaccharides (galactomannan), and cell wall components (β-D glucan) were selected by statistical filtering for patients with leukemia as a primary underlying diagnosis. Quantitative measurements were developed to qualify the differential expression of the candidate host response proteins using selective reaction monitoring mass spectrometry assays, and then applied to a separate cohort of 57 patients with leukemia. In this verification cohort, a machine learning ensemble-based algorithm, generalized pathseeker (GPS) produced a greater case classification accuracy than galactomannan (GM) or host proteins alone. In conclusion, Integration of host response proteins with GM improves the diagnostic detection of probable IPA in patients undergoing treatment for hematologic malignancy. Upon further validation, early detection of probable IPA in leukemia treatment will provide opportunities for earlier interventions and interventional clinical trials.

Use of surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) to study protein expression in a rat model of cocaine withdrawal.

Surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) is an analytical technology for proteomic analysis that combines chromatography and mass spectrometry. At present, this technology is most commonly being exploited for the simultaneous measurement of numerous proteins in serum, but has also been utilized in organ tissue, although rarely in the brain. We applied SELDI-TOF MS technology to study protein expression in the brain of rats withdrawn from repeated cocaine exposure. Our goals were to optimize sample preparation and ProteinChip Array protocols for brain tissue, to verify the reproducibility of SELDI-TOF mass spectra and to determine whether SELDI-TOF MS detects differentially expressed proteins in cocaine- versus saline-treated rats. Consequently, we have developed an optimal protocol and generated a reproducible spectral pattern with six dominant peaks in all test samples. We have detected two smaller peaks (m/z: 5179, 5030) that were significantly increased (p < 0.05) in cocaine-treated rats compared to saline-treated rats. In summary, the application of SELDI-TOF MS to the study of protein expression in a rat model of cocaine withdrawal is feasible and has the potential to generate new hypotheses.