Proteomic analysis reveals alterations in the renal kallikrein pathway during hypoxia-induced hypertension

Obstructive sleep apnea syndrome (OSAS), a disorder characterized by episodic hypoxia (EH) during sleep, is associated with systemic hypertension. We used proteomic analysis to examine differences in rat kidney protein expression during EH, and their potential relationship to EH-induced hypertension. Young male Sprague-Dawley rats were exposed to either EH or sustained hypoxia (SH) for 14 (EH14/SH14) and 30 (EH30/SH30) days. Mean arterial blood pressure was significantly increased only in EH30 (p < 0.0002). Kidney proteins were resolved by two-dimensional-PAGE and were identified by MALDI-MS. Renal expression of kallistatin, a potent vasodilator, was down-regulated in all animals. Expression of alpha-1-antitrypsin, an inhibitor of kallikrein activation, was up-regulated in EH but down-regulated in SH. Western blotting showed significant elevation of B(2)-bradykinin receptor expression in all normotensive animals but remained unchanged in hypertensive animals. Proteins relevant to vascular hypertrophy, such as smooth muscle myosin and protein-disulfide isomerase were up-regulated in EH30 but were down-regulated in SH30. These data indicate that EH induces changes in renal protein expression consistent with impairment of vasodilation mediated by the kallikrein-kallistatin pathway and vascular hypertrophy. In contrast, SH-induced changes suggest the kallikrein- and bradykinin-mediated compensatory mechanisms for prevention of hypertension and vascular remodeling. To test the hypothesis suggested by the proteomic data, we measured the effect of EH on blood pressure in transgenic hKLK1 rats that overexpress human kallikrein. Transgenic hKLK1 animals were protected from EH-induced hypertension. We conclude that EH-induced hypertension may result, at least in part, from altered regulation of the renal kallikrein system.     

Proteomic pathway analysis reveals inflammation increases myeloid-derived suppressor cell resistance to apoptosis

Myeloid-derived suppressor cells (MDSC) accumulate in patients and animals with cancer where they mediate systemic immune suppression and obstruct immune-based cancer therapies. We have previously demonstrated that inflammation, which frequently accompanies tumor onset and progression, increases the rate of accumulation and the suppressive potency of MDSC. To determine how inflammation enhances MDSC levels and activity we used mass spectrometry to identify proteins produced by MDSC induced in highly inflammatory settings. Proteomic pathway analysis identified the Fas pathway and caspase network proteins, leading us to hypothesize that inflammation enhances MDSC accumulation by increasing MDSC resistance to Fas-mediated apoptosis. The MS findings were validated and extended by biological studies. Using activated caspase 3 and caspase 8 as indicators of apoptosis, flow cytometry, confocal microscopy, and Western blot analyses demonstrated that inflammation-induced MDSC treated with a Fas agonist contain lower levels of activated caspases, suggesting that inflammation enhances resistance to Fas-mediated apoptosis. Resistance to Fas-mediated apoptosis was confirmed by viability studies of MDSC treated with a Fas agonist. These results suggest that an inflammatory environment, which is frequently present in tumor-bearing individuals, protects MDSC against extrinsic-induced apoptosis resulting in MDSC with a longer in vivo half-life, and may explain why MDSC accumulate more rapidly and to higher levels in inflammatory settings.     

Proteomic analysis reveals novel binding partners of metabotropic glutamate receptor 1

Regulated trafficking of neurotransmitter receptors is critical to normal neurodevelopment and neuronal signaling. Group I mGluRs (mGluR1/5 and their splice variants) are G protein-coupled receptors enriched at excitatory synapses, where they serve to modulate glutamatergic transmission. The mGluR1 splice variants mGluR1a and mGluR1b are broadly expressed in the central nervous system and differ in their signaling and trafficking properties. Several proteins have been identified that selectively interact with mGluR1a and participate in receptor trafficking but no proteins interacting with mGluR1b have thus far been reported. We have used a proteomic strategy to isolate and identify proteins that co-purify with mGluR1b in Madin-Darby Canine Kidney (MDCK) cells, an established model system for trafficking studies. Here, we report the identification of 10 novel candidate mGluR1b-interacting proteins. Several of the identified proteins are structural components of the cell cytoskeleton, while others serve as cytoskeleton-associated adaptors and motors or endoplasmic reticulum-associated chaperones. Findings from this work will help unravel the complex cellular mechanisms underlying mGluR trafficking under physiological and pathological conditions.     

Proteomic analysis on insulin signaling in human hematopoietic cells: identification of CLIC1 and SRp20 as novel downstream effectors of insulin

Insulin/IGF-I-dependent signals play important roles for the regulation of proliferation, differentiation, metabolism, and autophagy in various cells, including hematopoietic cells. Although the early protein kinase activation cascade has been intensively studied, the whole picture of intracellular signaling events has not yet been clarified. To identify novel downstream effectors of insulin-dependent signals in relatively early phases, we performed high-resolution two-dimensional electrophoresis (2-DE)-based proteomic analysis using human hematopoietic cells 1 h after insulin stimulation. We identified SRp20, a splicing factor, and CLIC1, an intracellular chloride ion channel, as novel downstream effectors besides previously reported effectors of Rho-guanine nucleotide dissociation inhibitor 2 and glutathione S-transferase-pi. Reduction in SRp20 was confirmed by one-dimensional Western blotting. Moreover, MG-132, a proteasome inhibitor, prevented this reduction. By contrast, upregulation of CLIC1 was not observed in one-dimensional Western blotting, unlike the 2-DE results. As hydrophilic proteins were predominantly recovered in 2-DE, the discrepancy between the 1-DE and 2-DE results may indicate a certain qualitative change of the protein. Indeed, the nuclear localization pattern of CLIC1 was remarkably changed by insulin stimulation. Thus insulin induces the proteasome-dependent degradation of SRp20 as well as the subnuclear relocalization of CLIC1.     

Proteomic analysis reveals novel binding partners of dysbindin, a schizophrenia-related protein

Schizophrenia is a complex mental disorder with fairly high level of heritability. Dystrobrevin binding protein 1, a gene encoding dysbindin protein, is a susceptibility gene for schizophrenia that was identified by family-based association analysis. Recent studies revealed that dysbindin is involved in the exocytosis and/or formation of synaptic vesicles. However, the molecular function of dysbindin in synaptic transmission is largely unknown. To investigate the signaling pathway in which dysbindin is involved, we isolated dysbindin-interacting molecules from rat brain lysate by combining ammonium sulfate precipitation and dysbindin-affinity column chromatography, and identified dysbindin-interacting proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Proteins involved in protein localization process, including Munc18-1, were identified as dysbindin-interacting proteins. Munc18-1 was co-immunoprecipitated with dysbindin from rat brain lysate, and directly interacted with dysbindin in vitro . In primary cultured rat hippocampal neurons, a part of dysbindin was co-localized with Munc18-1 at pre-synaptic terminals. Our result suggests a role for dysbindin in synaptic vesicle exocytosis via interaction with Munc18-1.     

Proteomic analysis of cellular signaling

Protein phosphorylation events are key regulators of cellular signaling processes. In the era of functional genomics, rational drug design programs demand large-scale high-throughput analysis of signal transduction cascades. Significant improvements in the area of mass spectrometry-based proteomics have provided exciting opportunities for rapid progress toward global protein phosphorylation analysis. This review summarizes several recent advances made in the field of phosphoproteomics with an emphasis placed on mass spectrometry instrumentation, enrichment methods and quantification strategies. In the near future, these technologies will provide a tool that can be used for quantitative investigation of signal transduction pathways to generate new insights into biologic systems.     

Proteomic analysis of endothelial lipid rafts reveals a novel role of statins in antioxidation

As inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, statins have pleiotropic vascular-protective effects, such as anti-inflammatory and antioxidative effects. We investigated the short-term beneficial effects of statins on modulating the translocation of lipid-raft-related proteins in endothelial cells (ECs). Human umbilical vein ECs were treated with atorvastatin for 30 min or 2 h; lipid-raft proteins were isolated and examined by quantitative proteome assay. Functional classification of identified proteins in lipid rafts revealed upregulated antioxidative proteins; downregulated proteins were associated with inflammation and cell adhesion. Among proteins verified by Western blot analysis, endoplasmic reticulum protein 46 (ERp46) showed increased level in lipid rafts with atorvastatin. Further, atorvastatin inhibited the activation of membrane-bound NADPH oxidase in both untreated and angiotensin II-treated ECs, as shown by reduced reactive oxygen species production. Co-immunoprecipitation and immunofluorescence experiments revealed that atorvastatin increased the association of ERp46 and Nox2, an NADPH oxidase isoform, in lipid rafts, thereby inhibiting Nox2 assembly with its regulatory subunits, such as p47phox and p67phox. Our results reveal a novel antioxidative role of atorvastatin by promoting the membrane translocation of ERp46 and its binding with Nox2 to inhibit Nox2 activity in ECs, which may offer another insight into the pleiotropic functions of statins.     

Proteomic analysis of cellular signaling

Protein phosphorylation events are key regulators of cellular signaling processes. In the era of functional genomics, rational drug design programs demand large-scale high-throughput analysis of signal transduction cascades. Significant improvements in the area of mass spectrometry-based proteomics have provided exciting opportunities for rapid progress toward global protein phosphorylation analysis. This review summarizes several recent advances made in the field of phosphoproteomics with an emphasis placed on mass spectrometry instrumentation, enrichment methods and quantification strategies. In the near future, these technologies will provide a tool that can be used for quantitative investigation of signal transduction pathways to generate new insights into biologic systems.     

Proteomic analysis of bovine sperm YWHA binding partners identify proteins involved in signaling and metabolism

Posttranslational modification of proteins by phosphorylation is involved in regulation of sperm function. Protein phosphatase 1 gamma isoform 2 (PPP1CC_v2) and protein YWHA (also known as 14-3-3) are likely to be key molecules in pathways involving sperm protein phosphorylation. We have shown that phosphorylated PPP1CC_v2 is bound to protein YWHAZ in spermatozoa. In somatic cells, protein YWHA is known to bind a number of phosphoproteins involved in signaling and energy metabolism. Thus, in addition to PPP1CC_v2, it is likely that sperm contain other YWHA-binding proteins. A goal of the present study was to identify these sperm YWHA-binding proteins. The binding proteins were isolated by affinity chromatography with GST-YWHAZ followed by elution with a peptide, R-11, which is known to disrupt YWHA complexes. The YWHA-binding proteins in sperm can be classified as those involved in fertilization, acrosome reaction, energy metabolism, protein folding, and ubiquitin-mediated proteolysis. A subset of these putative YWHA-binding proteins contain known amino acid consensus motifs, not only for YWHA binding but also for PPP1C binding. Identification of sperm PPP1CC_v2-binding proteins by microcystin-agarose chromatography confirmed that PPP1CC_v2 and YWHA interactomes contain several common proteins. These are metabolic enzymes phosphoglycerate kinase 2, hexokinase 1, and glucose phosphate isomerase; proteins involved in sperm-egg fusion; angiotensin-converting enzyme, sperm adhesion molecule, and chaperones; heat shock 70-kDa protein 5 (glucose-regulated protein 78 kDa; and heat shock 70-kDa protein 1-like. These proteins are likely to be phosphoproteins and potential PPP1CC_v2 substrates. Our data suggest that in addition to potential regulation of a number of important sperm functions, YWHA may act as an adaptor molecule for a subset of PPP1CC_v2 substrates.     

Proteomic and bioinformatic analysis of epithelial tight junction reveals an unexpected cluster of synaptic molecules

Background  

Zonula occludens, also known as the tight junction, is a specialized cell-cell interaction characterized by membrane "kisses" between epithelial cells. A cytoplasmic plaque of ~100 nm corresponding to a meshwork of densely packed proteins underlies the tight junction membrane domain. Due to its enormous size and difficulties in obtaining a biochemically pure fraction, the molecular composition of the tight junction remains largely unknown.     

Proteomic analysis of human substantia nigra identifies novel candidates involved in Parkinson's disease pathogenesis

Parkinson's disease (PD) pathology spreads throughout the brain following a region‐specific process predominantly affecting the substantia nigra (SN) pars compacta. SN exhibits a progressive loss of dopaminergic neurons responsible for the major cardinal motor symptoms, along with the occurrence of Lewy bodies in the surviving neurons. To gain new insights into the underlying pathogenic mechanisms in PD, we studied postmortem nigral tissues dissected from pathologically confirmed PD cases (n = 5) and neurologically intact controls (n = 8). Using a high‐throughput shotgun proteomic strategy, we simultaneously identified 1795 proteins with concomitant quantitative data. To date, this represents the most extensive catalog of nigral proteins. Of them, 204 proteins displayed significant expression level changes in PD patients versus controls. These were involved in novel or known pathogenic processes including mitochondrial dysfunction, oxidative stress, or cytoskeleton impairment. We further characterized four candidates that might be relevant to PD pathogenesis. We confirmed the differential expression of ferritin‐L and seipin by Western blot and demonstrated the neuronal localization of gamma glutamyl hydrolase and nebulette by immunohistochemistry. Our preliminary findings suggest a role for nebulette overexpression in PD neurodegeneration, through mechanisms that may involve cytoskeleton dynamics disruption. All MS data have been deposited in the ProteomeXchange with identifier PXD000427 ( http://proteomecentral.proteomexchange.org/dataset/PXD000427 ).     

Proteomic analysis reveals new cardiac-specific dystrophin-associated proteins

Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.     

Proteomic analysis reveals novel binding partners of MIP‐T3 in human cells

MIP‐T3 (microtubule‐interacting protein associated with TRAF3) is a microtubule‐interacting protein that evolutionarily conserved from worms to humans, but whose cellular functions remains unknown. To get insight into the functions of MIP‐T3, we set out to identify MIP‐T3 interacting proteins by immunoprecipitation in human embryonic kidney 293 cells and MS analysis. As the results, a total of 34 proteins were identified and most of them were novel MIP‐T3 putative partners. The MIP‐T3‐associated proteins could be grouped into nine clusters based on their molecule functions, including cytoskeleton, chaperone, nucleic acid binding, kinase and so on. Three MIP‐T3‐interacted proteins – actin, HSPA8 and tubulin – were further confirmed by reciprocal coimmunoprecipitations and colocalization analysis. The interaction of MIP‐T3 with both actin filaments and microtubule suggested that MIP‐T3 may play an important role in regulation of cytoskeleton dynamics in cells. Our results therefore not only uncover a large number of MIP‐T3‐associated proteins that possess a variety of cellular functions, but also provide new research directions for the study of the functions of MIP‐T3.     

Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii

Rhoptries are specialized secretory organelles that are uniquely present within protozoan parasites of the phylum Apicomplexa. These obligate intracellular parasites comprise some of the most important parasites of humans and animals, including the causative agents of malaria (Plasmodium spp.) and chicken coccidiosis (Eimeria spp.). The contents of the rhoptries are released into the nascent parasitophorous vacuole during invasion into the host cell, and the resulting proteins often represent the literal interface between host and pathogen. We have developed a method for highly efficient purification of rhoptries from one of the best studied Apicomplexa, Toxoplasma gondii, and we carried out a detailed proteomic analysis using mass spectrometry that has identified 38 novel proteins. To confirm their rhoptry origin, antibodies were raised to synthetic peptides and/or recombinant protein. Eleven of 12 of these yielded antibody that showed strong rhoptry staining by immunofluorescence within the rhoptry necks and/or their bulbous base. Hemagglutinin epitope tagging confirmed one additional novel protein as from the rhoptry bulb. Previously identified rhoptry proteins from Toxoplasma and Plasmodium were unique to one or the other organism, but our elucidation of the Toxoplasma rhoptry proteome revealed homologues that are common to both. This study also identified the first Toxoplasma genes encoding rhoptry neck proteins, which we named RONs, demonstrated that toxofilin and Rab11 are rhoptry proteins, and identified novel kinases, phosphatases, and proteases that are likely to play a key role in the ability of the parasite to invade and co-opt the host cell for its own survival and growth.     

Integrating novel signaling pathways involved in erythropoiesis

Many extrinsic and intrinsic factors control the development of red blood cells from committed progenitors, with the Erythropoietin-receptor (Epo-R) signaling network being the primary controlling molecular hub. Although much is understood about erythroid signaling pathways, new and intriguing factors that influence different aspects of erythroid cell development are still being uncovered. New extrinsic effectors include hypoxia and polymeric IgA1 (pIgA1), and new Epo-R signaling pathway components include Lyn/Cbp and Lyn/Liar. Hypoxia directly activates committed erythroid progenitors to expand, whereas pIgA1 activates the Akt and MAP-Kinase (MAPK) pathways through transferrin receptors on more mature erythroid cells. The Lyn/Cbp pathway controls the activity and protein levels of Lyn through recruitment of Csk and SOCS1, as well as feeding into the control of other pathways mediated by recruitment of ras-GAP, PI3-kinase, PLCγ, Fes, and EBP50. Nuclear/cytoplasmic shuttling of Lyn and other signaling molecules is influenced by Liar and results in regulation of their intersecting signaling pathways. The challenge of future research is to flesh out the details of these new signaling regulators/networks and integrate their influences during the different stages of erythropoiesis.