Bruce A. Stanley

Functional Proteomics of Arabidopsis thaliana Guard Cells Uncovers New Stomatal Signaling Pathways

We isolated a total of 3 × 108 guard cell protoplasts from 22,000 Arabidopsis thaliana plants and identified 1734 unique proteins using three complementary proteomic methods: protein spot identification from broad and narrow pH range two-dimensional (2D) gels, and 2D liquid chromatography–matrix assisted laser desorption/ionization multidimensional protein identification technology. This extensive single-cell-type proteome includes 336 proteins not previously represented in transcriptome analyses of guard cells and 52 proteins classified as signaling proteins by Gene Ontology analysis, of which only two have been previously assessed in the context of guard cell function. THIOGLUCOSIDE GLUCOHYDROLASE1 (TGG1), a myrosinase that catalyzes the production of toxic isothiocyanates from glucosinolates, showed striking abundance in the guard cell proteome. tgg1 mutants were hyposensitive to abscisic acid (ABA) inhibition of guard cell inward K+ channels and stomatal opening, revealing that the glucosinolate-myrosinase system, previously identified as a defense against biotic invaders, is required for key ABA responses of guard cells. Our results also suggest a mechanism whereby exposure to abiotic stresses may enhance plant defense against subsequent biotic stressors and exemplify how enhanced knowledge of the signaling networks of a specific cell type can be gained by proteomics approaches.

Proteomics of cauda epididymal fluid from mature Holstein bulls.

The proteome of cauda epididymal fluid (CEF) from Holstein bulls was defined. Fluid was collected from the vas deferens, subjected to 2-D SDS-PAGE and spots identified by CapLC-MS/MS and MALDI-ToF/ToF. Because albumin accounted for 21.1% of all spot intensities in the gels examined by PDQuest, samples were subjected to albumin depletion and then analyzed again as before. Original CEF gels had 114 ± 3 spots, including as the most abundant: albumin, epididymal secretory protein E1, prostaglandin d-synthase and gelsolin. Epididymal fluid also expressed: clusterin, transferrin, N-acetyl-β-glucosaminidase, cauxin, glutathione peroxidase, acidic seminal fluid protein (aSFP), aldehyde reductase, α-l-fucosidase, α-1-β-glycoprotein, apolipoprotein A-1, β actin, calmodulin, cathepsin D, cystatin E/M, enolase, galectin 3-binding protein, leucine amino-peptidase and nucleobindin. Albumin depletion decreased that very spot to 10% of its original intensity and the resulting gels had, on average, 137 ± 4 spots. Spots identified as dipeptidyl-peptidase 7, angiotensin-converting enzyme, arylsulfatase A, aspartylglucosaminidase, serine protease inhibitors, new isoforms of calmodulin, cystatin E/M and a 17-kDa nucleobindin appeared only in depleted maps. This study is the first to report nucleobindin and aSFP as epididymal components. We suggest that CEF proteins act to facilitate membrane remodeling, transport of lipophilic substances, protect sperm and prevent premature acrosome reaction.

Integrated quantitative analysis of nitrogen stress response in Chlamydomonas reinhardtii using metabolite and protein profiling.

Nitrogen starvation induces a global stress response in microalgae that results in the accumulation of lipids as a potential source of biofuel. Using GC-MS-based metabolite and iTRAQ-labeled protein profiling, we examined and correlated the metabolic and proteomic response of Chlamydomonas reinhardtii under nitrogen stress. Key amino acids and metabolites involved in nitrogen sparing pathways, methyl group transfer reactions, and energy production were decreased in abundance, whereas certain fatty acids, citric acid, methionine, citramalic acid, triethanolamine, nicotianamine, trehalose, and sorbitol were increased in abundance. Proteins involved in nitrogen assimilation, amino acid metabolism, oxidative phosphorylation, glycolysis, TCA cycle, starch, and lipid metabolism were elevated compared with nonstressed cultures. In contrast, the enzymes of the glyoxylate cycle, one carbon metabolism, pentose phosphate pathway, the Calvin cycle, photosynthetic and light harvesting complex, and ribosomes were reduced. A noteworthy observation was that citrate accumulated during nitrogen stress coordinate with alterations in the enzymes that produce or utilize this metabolite, demonstrating the value of comparing protein and metabolite profiles to understand complex patterns of metabolic flow. Thus, the current study provides unique insight into the global metabolic adjustments leading to lipid storage during N starvation for application toward advanced biofuel production technologies.

PROCESSING OF MAMMALIAN AND PLANT S-ADENOSYLMETHIONINE DECARBOXYLASE PROENZYMES

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl enzyme, and the pyruvate is formed in an intramolecular reaction that cleaves a proenzyme precursor and converts a serine residue into pyruvate. The wild type potato AdoMetDC proenzyme processed much faster than the human proenzyme and did not require putrescine for an optimal rate of processing despite the presence of three acidic residues (equivalent to Glu11, Glu178, and Glu256) that were demonstrated in previous studies to be required for the putrescine activation of human AdoMetDC proenzyme processing (Stanley, B. A., Shantz, L. M., and Pegg, A. E. (1994) J. Biol. Chem. 269, 7901–7907). A fourth residue that is also needed for the putrescine stimulation of human AdoMetDC proenzyme processing was identified in the present studies, and this residue (Asp174) is not present in the potato sequence. The site of potato AdoMetDC proenzyme processing was found to be Ser73 in the conserved sequence, YVLSESS, which is the equivalent of Ser68 in the human sequence. Replacement of the serine precursor with threonine or cysteine by site-directed mutagenesis in either the potato or the human AdoMetDC proenzyme did not prevent processing but caused a significant reduction in the rate. Although the COOH-terminal regions of the known eukaryotic AdoMetDCs are not conserved, only relatively small truncations of 8 residues from the human protein and 25 residues from the potato proenzyme were compatible with processing. The maximally truncated proteins show no similarity in COOH-terminal amino acid sequence but each contained 46 amino acid residues after the last conserved sequence, suggesting that the length of this section of the protein is essential for maintaining the proenzyme conformation needed for autocatalytic processing.

ABA-regulated G protein signaling in Arabidopsis guard cells: a proteomic perspective.

Signaling cascades mediated by heterotrimeric G proteins are ubiquitous and important signal transduction mechanisms in both metazoans and plants. In the model plant Arabidopsis thaliana, the sole canonical G protein alpha subunit, GPA1, has been implicated in multiple signaling events, including guard cell movement regulated by the plant stress hormone abscisic acid (ABA). However, only a handful of proteins have been demonstrated to be involved in GPA1 signaling to date. Here, we compared the proteome composition of guard cells from wild type Col vs gpa1-4 null mutants with and without ABA treatment using iTRAQ technology to identify guard cell proteins whose abundance was affected by ABA and/or GPA1. After imposition of strict selection criteria, the abundance of two proteins in Col and six proteins in gpa1-4 was found to be affected by ABA in guard cells, and 18 guard cell proteins were quantitatively affected by the mutation of GPA1. On the basis of known functions of the differentially expressed proteins, our data suggest that GPA1 inhibits guard cell photosynthesis and promotes the availability of reactive oxygen species (ROS) in guard cells. These results exemplify how iTRAQ can be used to quantitatively study single cell signaling pathways in Arabidopsis.

The crystal structure of human S-adenosylmethionine decarboxylase at 2.25 A resolution reveals a novel fold.

BACKGROUND S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical regulatory enzyme of the polyamine synthetic pathway, and a well-studied drug target. The AdoMetDC decarboxylation reaction depends upon a pyruvoyl cofactor generated via an intramolecular proenzyme self-cleavage reaction. Both the proenzyme-processing and substrate-decarboxylation reactions are allosterically enhanced by putrescine. Structural elucidation of this enzyme is necessary to fully interpret the existing mutational and inhibitor-binding data, and to suggest further experimental studies. RESULTS The structure of human AdoMetDC has been determined to 2.25 A resolution using multiwavelength anomalous diffraction (MAD) phasing methods based on 22 selenium-atom positions. The quaternary structure of the mature AdoMetDC is an (alpha beta)2 dimer, where alpha and beta represent the products of the proenzyme self-cleavage reaction. The architecture of each (alpha beta) monomer is a novel four-layer alpha/beta-sandwich fold, comprised of two antiparallel eight-stranded beta sheets flanked by several alpha and 3(10) helices. CONCLUSIONS The structure and topology of AdoMetDC display internal symmetry, suggesting that this protein may be the product of an ancient gene duplication. The positions of conserved, functionally important residues suggest the location of the active site and a possible binding site for the effector molecule putrescine.

Identification and Analysis of Occludin Phosphosites: A Combined Mass Spectrometry and Bioinformatics Approach

The molecular function of occludin, an integral membrane component of tight junctions, remains unclear. VEGF-induced phosphorylation sites were mapped on occludin by combining MS data analysis with bioinformatics. In vivo phosphorylation of Ser490 was validated and protein interaction studies combined with crystal structure analysis suggest that Ser490 phosphorylation attenuates the interaction between occludin and ZO-1. This study demonstrates that combining MS data and bioinformatics can successfully identify novel phosphorylation sites from limiting samples.

Apolipoprotein A-IV, a Putative Satiety/Antiatherogenic Factor, Rises After Gastric Bypass

Roux‐en‐Y gastric bypass surgery (RYGBP) leads to improvements in satiety and obesity‐related comorbidities. The mechanism(s) underlying these improvements are not known but may be revealed in part by discovery proteomics. Therefore, fasting plasma was collected from 12 subjects (mean BMI >45) during RYGBP and during a second procedure ∼17 months later. Body weight, obesity‐related comorbidities, and medication use were decreased after RYGBP. Mass spectrometry‐based proteomic analysis was performed on a subset of seven samples using isobaric isotope‐coded affinity tags (four plex iTRAQ). Initial proteomic analysis (n = 7) quantified and identified hundreds of plasma proteins. Manual inspection of the data revealed a 2.6 ± 0.5‐fold increase in apolipoprotein A‐IV (apo A‐IV, gene designation: APOA4), a ∼46‐kDa glycoprotein synthesized mainly in the bypassed small bowel and liver after RYGBP. The change in apo A‐IV was significantly greater than other apolipoproteins. Immunoblot analysis of the full longitudinal sample set (n = 12) indicated even higher increases (8.3 ± 0.2 fold) in apo A‐IV. Thus iTRAQ may underestimate the changes in protein concentrations compared to western blotting of apo A‐IV. Apo A‐IV inhibits gastric emptying and serves as a satiety factor whose synthesis and secretion are increased by the ingestion of dietary fat. It also possesses anti‐inflammatory and antiatherogenic properties. Based on these functions, we speculate changes in apo A‐IV may contribute to weight loss as well as the improvements in inflammation and cardiovascular disease after RYGBP. In addition, the findings provide evidence validating the use of iTRAQ proteomics in discovery‐based studies of post‐RYGBP improvements in obesity‐related medical comorbidities.

Proteomic profiling of human plasma by iTRAQ reveals down-regulation of ITI-HC3 and VDBP by cigarette smoking.

Biomarkers in noninvasive fluids indicative of cigarette smokes effects are urgently needed. In this pilot study, we utilized the proteomic approach, isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to identify differentially expressed plasma proteins in healthy cigarette smokers compared to healthy nonsmokers; select proteins were further confirmed by immunoblot analysis. Significant, differentially expressed proteins identified in the plasma separated subjects based on their condition as smokers or nonsmokers. Several of the proteins identified in this study are associated with immunity and inflammatory responses and have been shown to be associated with tobacco-related diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. Proteins up-regulated in smokers included complement component 8 polypeptide chains α, β, and γ, and mannose-binding protein C, and proteins down-regulated included inter-α-trypsin inhibitor heavy chain H3 (ITI-HC3) and vitamin D-binding protein (VDBP). In addition, gelsolin and vitronectin, known tissue leakage proteins, were up- and down-regulated, respectively. Our results demonstrate for the first time that chronic cigarette smoking can influence the expression profile of the human plasma proteome. Proteins identified in this pilot study may serve as candidate biomarkers of diseases resulting from exposure to cigarette smoke in future molecular epidemiological studies.

Immunodepletion Plasma Proteomics by TripleTOF 5600 and Orbitrap Elite/LTQ-Orbitrap Velos/Q Exactive Mass Spectrometers

Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered >250 GB of data in 2 months. Several analysis algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.