The Arabidopsis Leucine-Rich Repeat Receptor Kinase BIR3 Negatively Regulates BAK1 Receptor Complex Formation and Stabilizes BAK1.

BAK1 is a coreceptor and positive regulator of multiple ligand binding leucine-rich repeat receptor kinases (LRR-RKs) and is involved in brassinosteroid (BR)-dependent growth and development, innate immunity, and cell death control. The BAK1-interacting LRR-RKs BIR2 and BIR3 were previously identified by proteomics analyses of in vivo BAK1 complexes. Here, we show that BAK1-related pathways such as innate immunity and cell death control are affected by BIR3 in Arabidopsis thaliana BIR3 also has a strong negative impact on BR signaling. BIR3 directly interacts with the BR receptor BRI1 and other ligand binding receptors and negatively regulates BR signaling by competitive inhibition of BRI1. BIR3 is released from BAK1 and BRI1 after ligand exposure and directly affects the formation of BAK1 complexes with BRI1 or FLAGELLIN SENSING2. Double mutants of bak1 and bir3 show spontaneous cell death and constitutive activation of defense responses. BAK1 and its closest homolog BKK1 interact with and are stabilized by BIR3, suggesting that bak1 bir3 double mutants mimic the spontaneous cell death phenotype observed in bak1 bkk1 mutants via destabilization of BIR3 target proteins. Our results provide evidence for a negative regulatory mechanism for BAK1 receptor complexes in which BIR3 interacts with BAK1 and inhibits ligand binding receptors to prevent BAK1 receptor complex formation.

Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle.

Plants constantly adjust their repertoire of plasma membrane proteins that mediates transduction of environmental and developmental signals as well as transport of ions, nutrients, and hormones. The importance of regulated secretory and endocytic trafficking is becoming increasingly clear; however, our knowledge of the compartments and molecular machinery involved is still fragmentary. We used immunogold electron microscopy and confocal laser scanning microscopy to trace the route of cargo molecules, including the BRASSINOSTEROID INSENSITIVE1 receptor and the REQUIRES HIGH BORON1 boron exporter, throughout the plant endomembrane system. Our results provide evidence that both endocytic and secretory cargo pass through the trans-Golgi network/early endosome (TGN/EE) and demonstrate that cargo in late endosomes/multivesicular bodies is destined for vacuolar degradation. Moreover, using spinning disc microscopy, we show that TGN/EEs move independently and are only transiently associated with an individual Golgi stack.

Identification of in vitro phosphorylation sites in the Arabidopsis thaliana somatic embryogenesis receptor-like kinases.

The Arabidopsis thaliana somatic embryogenesis receptor-like kinase (SERK) family consists of five leucine-rich repeat receptor-like kinases (LRR-RLKs) with diverse functions such as brassinosteroid insensitive 1 (BRI1)-mediated brassinosteroid perception, development and innate immunity. The autophosphorylation activity of the kinase domains of the five SERK proteins was compared and the phosphorylated residues were identified by LC-MS/MS. Differences in autophosphorylation that ranged from high activity of SERK1, intermediate activities for SERK2 and SERK3 to low activity for SERK5 were noted. In the SERK1 kinase the C-terminally located residue Ser-562 controls full autophosphorylation activity. Activation loop phosphorylation, including that of residue Thr-462 previously shown to be required for SERK1 kinase activity, was not affected. In vivo SERK1 phosphorylation was induced by brassinosteroids. Immunoprecipitation of CFP-tagged SERK1 from plant extracts followed by MS/MS identified Ser-303, Thr-337, Thr-459, Thr-462, Thr-463, Thr-468, and Ser-612 or Thr-613 or Tyr-614 as in vivo phosphorylation sites of SERK1. Transphosphorylation of SERK1 by the kinase domain of the main brassinosteroid receptor BRI1 occurred only on Ser-299 and Thr-462. This suggests both intra- and intermolecular control of SERK1 kinase activity. Conversely, BRI1 was transphosphorylated by the kinase domain of SERK1 on Ser-887. BRI1 kinase activity was not required for interaction with the SERK1 receptor in a pull down assay.

The proteome of the human neuroblastoma cell line SH-SY5Y: an enlarged proteome.

The human neuroblastoma cell line SH-SY5Y (ATCC: CRL-2266) is widely used as a neural cellular model system. The hitherto existing proteome data (115 proteins) are here extended. A total of 1103 unique proteins of this cell line were identified using 2D-LC combined with MALDI-TOF/TOF-MS, SDS-PAGE with nano-LC-MS/MS, N-terminal COFRADIC analysis with nano-LC-MS/MS and 2D-PAGE with MALDI-TOF/TOF-MS peptide mass fingerprinting. The obtained proteome profile of this cell line is discussed.

Exploring natural genetic variation in tomato sucrose synthases on the basis of increased kinetic properties.

Sucrose synthase (SuSy) is one key enzyme directly hydrolyzing sucrose to supply substrates for plant metabolism, and is considered to be a biomarker for plant sink strength. Improvement in plant sink strength could lead to enhanced plant growth and yield. Cultivated tomatoes are known to have a narrow genetic diversity, which hampers further breeding for novel and improved traits in new cultivars. In this study, we observed limited genetic variation in SuSy1, SuSy3 and SuSy4 in 53 accessions of cultivated tomato and landraces, but identified a wealth of genetic diversity in 32 accessions of related wild species. The variation in the deduced amino acid sequences was grouped into 23, 22, and 17 distinct haplotypes for SuSy1/3/4, respectively. Strikingly, all known substrate binding sites were highly conserved, as well as most of the phosphorylation sites except in SuSy1. Two SuSy1 and three SuSy3 protein variants were heterologously expressed to study the effect of the amino acid changes on enzyme kinetic properties, i.e. maximal sucrose hydrolyzing capacity (Vmax), affinity for sucrose (Km), and catalytic efficiency (Vmax/Km) at 25°C and 16°C. SuSy1-haplotype#3 containing phosphorylation site Ser-16 did not have an improvement in the kinetic properties compared to the reference SuSy1-haplotype#1 containing Arg-16. Meanwhile SuSy3-haplotype#9 from a wild accession, containing four amino acid changes S53A, S106I, E727D and K741E, showed an increase in Vmax/Km at 16°C compared to the reference SuSy3-haplotype#1. This study demonstrates that SuSy kinetic properties can be enhanced by exploiting natural variation, and the potential of this enzyme to improve sucrose metabolism and eventually sink strength in planta.

Identification of Brassinosteroid Signaling Complexes by Coimmunoprecipitation and Mass Spectrometry.

A combination of coimmunoprecipitation (coIP) of tagged proteins followed by protein identification and quantitation using Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LCMS/MS) has proven to be a reliable method to qualitatively characterize membrane-bound receptor complexes from plants. Success depends on a range of parameters, such as abundance and stability of the complex and functionality of the tagged receptors, efficiency of the protein complex isolation procedure, MS equipment, and analysis software in use. In this Chapter, we focus on the use of one of the green fluorescent protein-tagged receptors of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, of which SERK3, also known as BRASSINOSTEROID INSENSITIVE1 (BRI1) ASSOCIATED KINASE1 (BAK1), is a coreceptor of BRI1. Like BRI1 itself, SERK3 is a leucine-rich repeat receptor kinase (LRR RK) with a single-pass transmembrane domain. The latest updated laboratory protocol is presented as well as examples of data analysis and typical results obtained. Potential drawbacks of the procedure employed for plant membrane proteins will be pointed out.

A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: structural determinants of redox potential.

Flavodoxin II from Azotobacter vinelandii is a "long-chain" flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structure determined to a resolution of 2.25 A by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five-stranded parallel beta-sheet flanked on either side by alpha-helices. An eight-residue insertion, compared with other long-chain flavodoxins, forms a short 3(10) helix preceding the start of the alpha3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent-accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E2 value in particular.

Intriguing mass spectrometric behavior of guanosine under low energy collision-induced dissociation: H2O adduct formation and gas-phase reactions in the collision cell.

An in-depth study of the fragmentation pathway of guanosine was conducted by using an in-source collision-induced dissociation high-mass accuracy tandem mass spectrometry experiment. The equivalent of MS4 data, a level of information normally achieved on ion trap instruments, was obtained on a Q-TOF mass spectrometer. The combination of the features of high-resolution, accuracy, and in-source CID permitted the unambiguous elucidation of the different fragmentation pathways. Furthermore the elemental compositions of the product ions generated were assigned and their mutual genealogical relationships established. Formerly proposed dissociation pathways of guanosine were revisited and elaborated on more deeply. Furthermore, the presence of H2O in the collision cell of several tandem MS instruments was demonstrated and its effect on the product ion spectra investigated. The neutral gain of H2O by particular fragments of guanosine was experimentally proven by using argon, saturated with H2(18)O, as the collision gas. Data indicating the occurrence of more complex reactions in the collision cell as a result of the presence of H2O were produced, specifically relating to neutral gain/neutral loss sequences. In silico calculations supported the experimental observation of neutral gain by guanosine fragments and predicted a similar behavior for adenosine. The latter was subsequently experimentally confirmed.

Search for substrates for prolyl oligopeptidase in porcine brain.

The function of prolyl oligopeptidase (PO) has been associated with several disorders of the central nervous system. The purpose of this study was to identify endogenous substrates for recombinant porcine PO in porcine brain. The smaller polypeptides were extracted from total brain homogenates and fractionated by two-dimensional chromatography prior to incubation with PO. Shifts in the mass spectrum between the control and the incubated sample, marked potential substrates. Using MSMS peptide sequencing techniques, we identified several fragments of intracellular proteins as potential substrates, which opens new perspectives for finding the function of PO in the intracellular space.

Molecular basis for redox-Bohr and cooperative effects in cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774: crystallographic and modeling studies of oxidized and reduced high-resolution structures at pH 7.6.

The tetraheme cytochrome c3 is a small metalloprotein with ca. 13,000 Da found in sulfate-reducing bacteria, which is believed to act as a partner of hydrogenase. The three-dimensional structure of the oxidized and reduced forms of cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774 at pH 7.6 were determined using high-resolution X-ray crystallography and were compared with the previously determined oxidized form at pH 4.0. Theoretical calculations were performed with both structures, using continuum electrostatic calculations and Monte Carlo sampling of protonation and redox states, in order to understand the molecular basis of the redox-Bohr and cooperativity effects related to the coupled transfer of electrons and protons. We were able to identify groups that showed redox-linked conformational changes. In particular, Glu61, His76, and propionate D of heme II showed important contributions to the redox-cooperativity, whereas His76, propionate A of heme I, and propionate D of heme IV were the key residues for the redox-Bohr effect. Upon reduction, an important movement of the backbone region surrounding hemes I and II was also identified, that, together with a few redox-linked conformational changes in side-chain residues, results in a significant decrease in the solvent accessibility of hemes I and II.

Analysis of DNA-phosphate adducts in vitro using miniaturized LC-ESI-MS/MS and column switching: phosphotriesters and alkyl cobalamins.

DNA-phosphate adducts are known to be formed by a variety of alkylating agents. Due to little or no repair of DNA-phosphate adducts, these adducts may offer increased possibilities of both identifying and quantifying DNA adducts. The formation of DNA-phosphate adducts leads to a complete esterification of the phosphate group giving rise to a phosphotriester configuration. This work consists of the characterization of ethyl phosphotriesters (Ethyl PTE) using miniaturized LC-ESI-MS/MS and column switching in enzymatic hydrolysate of DNA treated in vitro with the model compound N-ethyl-N-nitrosourea (ENU). In vitro ENU-treated DNA was enzymatically degraded using nuclease P1, phosphodiesterase, and alkaline phosphatase. The use of column switch allowed for large-volume injections, where unmodified nucleosides were discarded in the loading step. The analytes were forward flushed to the analytical column in the eluting step and separated using a linear gradient. Ten different ethyl PTEs (dGpEtdG, dApEtdA, dCpEtdC, TpEtT, dGpEtdA, dGpEtdC, dGpEtT, dApEtdC, dApEtT, and dCpEtT) were characterized by their masses and CAD product ion spectra. Measurements of accurate masses were carried out yielding experimental masses within 5 ppm of the calculated masses for 9 of the 10 ethyl PTEs. For comparison, the enzymatic hydrolysate of ENU-treated DNA was subjected to transalkylation of the DNA-phosphate adducts by cob(I)alamin. Formed ethyl-cobalamins were analyzed according to earlier developed methods. The limit of detection of an alkyl-cobalamin standard and an alkyl PTE standard was 2 fmol and 5 fmol, respectively.

Structural characterization of melphalan modified 2'-oligodeoxynucleotides by miniaturized LC-ES MS/MS.

In this study a miniaturized LC coupled to electrospray tandem mass spectrometry was used to analyze modifications originating from the interaction between the chemotherapeutic agent melphalan and 2'-oligodeoxynucleotides. Low energy CAD product ion spectra gave information about the specificity of melphalan alkylation with regard to certain DNA sequences. These data can be very useful to estimate the risk in the development of secondary leukaemia as a result of a melphalan cure. In the study of the interaction between melphalan and d(GG), differentiation could be made between alkylation on the 5'-side and alkylation on the 3'-side, because of the presence or absence of the alkylated w1 fragment in the low energy CAD spectra. In the other di-mers alkylation specificity for the different bases could be observed. Melphalan alkylation occurs in the sequence G > A > C > T. The study of the alkylated d(GGGG) revealed the presence of mainly 5'-end alkylation. Furthermore studies were performed which investigated other melphalan treated di-, tetra-, hepta-, and octa-mers.

Biochemical effect evaluation of perfluorooctane sulfonic acid-contaminated wood mice (Apodemus sylvaticus).

Wood mice (Apodemus sylvaticus) were captured at Blokkersdijk, a nature reserve in the immediate vicinity of a fluorochemical plant in Antwerp, Belgium, and at Galgenweel, 3 kilometers farther away. The liver perfluorooctane sulfonic acid (PFOS) concentrations in the Blokkersdijk mice were extremely high (0.47-178.55 micro g/g wet weight). Perfluorononanoic, perfluorodecanoic, perfluoroundecanoic, and perfluorododecanoic acids were found sporadically in the liver tissue of the Blokkersdijk mice. The liver PFOS concentrations at Galgenweel were significantly lower than those at Blokkersdijk (0.14-1.11 micro g/g wet weight). Further results suggest sex independence of the liver PFOS levels, increased levels of PFOS bioaccumulation in older mice, and maternal PFOS transfer to the young. Several liver end points were significantly elevated in the Blokkersdijk mice: liver weight, relative liver weight, peroxisomal beta-oxidation activity, microsomal lipid peroxidation level, and mitochondrial fraction protein content. For the mitochondrial fraction catalase activity, no significant difference between locations was found. The liver weight, relative liver weight, and liver microsomal lipid peroxidation level increased significantly with the liver PFOS concentration. No indications for PFOS-mediated effects on the serum triglyceride, cholesterol, or potassium levels were obtained. The liver PFOS concentration was negatively related to the serum alanine aminotransferase activity.

Qualitative study of in vivo melphalan adduct formation in the rat by miniaturized column-switching liquid chromatography coupled with electrospray mass spectrometry.

In a general study of DNA adduct formation with melphalan, rats were intravenously injected with a single high dose (10 mg kg(-1)). Adduct formation was studied at the nucleoside level in the target organs liver, bone marrow, kidney and blood with the use of 2D liquid chromatography/tandem mass spectrometry (LC/MS/MS). Adducts of dGuo and dAdo were detected under selected reaction monitoring in liver and bone marrow 10 h after administration of melphalan. In the DNA hydrolysates from kidney and blood a Gua-melphalan adduct was found, although in very low abundance. These first results of the search for in vivo-formed melphalan adducts in the rat showed that our miniaturized LC/MS technique is useful for investigating this type of compound. More experiments will be performed in this area to gather more information about the pharmacokinetics and the quantity of adducts formed.

Investigation of the use of immobilised metal affinity chromatography for the on-line sample clean up and pre-concentration of nucleotides prior to their determination by ion pair liquid chromatography-electrospray mass spectrometry: a pilot study.

This study explored an alternative way to enrich and pre-purify biological samples containing nucleoside mono-, di- and triphosphates. These compounds were trapped by immobilised metal affinity chromatography (IMAC) on a Poros 20 MC IMAC-column, which was conditioned with Fe3+. The IMAC-column was implemented in a column switching set-up separating nucleoside mono-, di- and triphosphates on a Hypersil ODS 35 mm x 0.3 mm capillary column hyphenated to electrospray mass spectrometry resulting in the first miniaturised column switching liquid chromatography-mass spectrometry (LC-MS) system for nucleotides.

Alkylation of DNA by melphalan: investigation of capillary liquid chromatography-electrospray ionization tandem mass spectrometry in the study of the adducts at the nucleoside level.

Nitrogen mustards are among the oldest cancer chemotherapeutic agents and remain the drugs of choice for treatment of many human cancers. A serious complication of treatment with nitrogen mustards is the increased risk of a secondary leukaemia in long-term survivors because not all alkylating agent interactions with DNA result in cell death. In an earlier study 2'-deoxy-5'-mononucleotide/melphalan adducts have been analysed by us by LC-ES MSMS. In this work we want to present the first results of the analysis of the corresponding 2'-deoxynucleoside/melphalan adducts from DNA hydrolysates by column switching/capillary LC-ES tandem mass spectrometry. Nucleosides, compared to nucleotides, give better chromatographic results and show a good sensitivity under electrospray (+) [ES(+)] ionisation. Several adducts were identified under ES(+) conditions. Mono-alkylated nucleoside adducts alkylated at the base moiety were identified for dGuo, dCyd and dAdo. Structures were identified by recording the low-energy CAD product ion scans. Also a mono-alkylated nucleotide pdA with alkylation position at the phosphate moiety could be detected. This proves that in the case of phosphate alkylation the enzymatic dephosphorylation reaction was inhibited. A Jurkat cell suspension was treated with melphalan (1 mM) and incubated at 37 degrees C (5% CO(2)). After 6 and 48 h, the DNA was isolated and enzymatically hydrolysed. The corresponding nucleoside pool was evaluated with the developed LC-MS method. In the 48-h experiment, one adduct could be identified as a N-7 alkylated dGuo. In the 6-h experiment, no adducts could be found. Additional experiments were done wherein Jurkat-DNA, isolated from a non-treated cell culture, was treated with melphalan. These results were analogous with the data found in melphalan-treated calf thymus DNA. Additionally, we tried to determine the exact alkylation position by interpreting high-resolution fragmentation spectra.

Determination of diazepam in aquatic samples by capillary liquid chromatography-electrospray tandem mass spectrometry.

In recent years growing attention has been paid toward the discharge, presence and potential adverse effects of pharmaceuticals in the environment. Using different existing analytical methods several studies have already identified a variety of drugs in waste-, surface- and drinking water. The monitoring of surface waters for drugs is of great importance because drugs are designed to be biological very active substances. A capillary LC/ES-MS-MS method has been developed that enables the sensitive and specific detection of diazepam in water samples up to 0.1 ng/ml (LOD). It requires neither multiple extraction steps, nor the use of large volumes of organic solvent. Applying this assay we have detected diazepam in 'in/effluent samples' collected in Belgium and demonstrated the applicability for water analysis without off-line pre-concentration of the analyte.

Exposure patterns of perfluorooctane sulfonate in aquatic invertebrates from the Western Scheldt estuary and the southern North Sea.

Over the past decades little research has been conducted on the environmental behavior and effects of fluorinated organochemicals (FOCs). Recently it has been reported that perfluorooctane sulfonic acid (PFOS) is occurring worldwide. Little is known about the PFOS levels in organisms originating from the southern North Sea and the Western Scheldt estuary. In this study, we determined, for the first time, the PFOS-exposure levels in Crangon crangon, Carcinus maenas, and Asterias rubens from these ecosystems. Concentrations on a wet-weight basis in soft tissues of shrimp, crab, and starfish ranged from 19 to 520 ng/g, from 24 to 877 ng/g, and from 9 to 176 ng/g, respectively. These results show the existence of a PFOS pollution gradient in organisms along the Western Scheldt estuary, with the highest concentrations near Antwerp. The range of PFOS levels in shrimp and crab are slightly higher in coastal regions compared with sampling sites in open water. This study shows widespread distribution of PFOS in the Belgian and Dutch marine and estuarine environment at rather high concentrations.

Perfluorooctane sulfonic acid in bib (Trisopterus luscus) and plaice (Pleuronectes platessa) from the Western Scheldt and the Belgian North Sea: distribution and biochemical effects.

A biomonitoring campaign was conducted in the Belgian North Sea and in the Western Scheldt (The Netherlands) with the primary goal to assess perfluorooctane sulfonic acid (PFOS) contamination and distribution in different biota. This study covers the results obtained for bib (Trisopterus luscus) and plaice (Pleuronectes platessa) and includes the assessment of some stress-related biochemical endpoints. Analysis of liver and muscle PFOS concentrations of both species provided evidence for the existence of a PFOS pollution gradient along the Western Scheldt with higher levels at the upstream locations and a lower degree of PFOS pollution at the marine locations. Cellular necrosis was studied by measuring aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the serum. Serum ALT but not serum AST was shown to correlate positively with the PFOS liver concentration in bib (r = 0.44, p < 0.05), indicating that PFOS might contribute to the induction of hepatic damage in bib in the area of study. Analysis of total carbohydrate, lipid, and protein content of bib liver tissue revealed a positive correlation between the protein content and the PFOS liver concentration (r = 0.55, p < 0.01). Whether this is due to induction of compensatory mechanisms, detoxification, or repair processes remains unclear.

Proteomics-based identification of low-abundance signaling and regulatory protein complexes in native plant tissues.

Owing to the low abundance of signaling proteins and transcription factors, their protein complexes are not easily identified by classical proteomics. The isolation of these protein complexes from endogenous plant tissues (rather than plant cell cultures) is therefore an important technical challenge. Here, we describe a sensitive, quantitative proteomics-based procedure to determine the composition of plant protein complexes. The method makes use of fluorophore-tagged protein immunoprecipitation (IP) and label-free mass spectrometry (MS)-based quantification to correct for nonspecifically precipitated proteins. We provide procedures for the isolation of membrane-bound receptor complexes and transcriptional regulators from nuclei. The protocol consists of an IP step (~6 h) and sample preparation for liquid chromatography-tandem MS (LC-MS/MS; 2 d). We also provide a guide for data analysis. Our single-step affinity purification protocol is a good alternative to two-step tandem affinity purification (TAP), as it is shorter and relatively easy to perform. The data analysis by label-free quantification (LFQ) requires a cheaper and less challenging experimental setup compared with known labeling techniques in plants.