Novel C-Raf phosphorylation sites: serine 296 and 301 participate in Raf regulation

The C-Raf kinase is regulated by numerous phosphorylation steps. To quantify the most prominent phosphorylation sites of C-Raf, we performed mass spectrometry analysis of wild-type C-Raf and the constitutively active C-Raf mutant C-Raf-Y340D/Y341D. We confirmed phosphorylation of most of the sites reported in the literature with the exception that we did not detect phosphorylation of threonine 268/269 (autophosphorylation sites) and threonine 491/serine 494 (kinase activation loop). Importantly, we detected novel phosphorylation sites at the positions of serine 296 and 301. The degree of phosphorylation in these positions depends on the level of activation of C-Raf. Furthermore, we show here, using point mutant forms of C-Raf kinases with serine to alanine and serine to aspartic acid substitution, that serines 296 and 301 contribute to negative regulation of C-Raf.     

The monoterpenoids citral and geraniol are moderate inhibitors of CYP2B6 hydroxylase activity

Monoterpenes are found in the volatile essence of flowers, plants oils, and herbal medicines. Some are commonly used as food additives and fragrance components, and many are found in cosmetics, soaps, cleaning products, disinfectants, preservatives, and medicines. We have recently discovered a moderate inhibitory effect of borneol and isoborneol toward CYP2B6-catalyzed bupropion hydroxylase activity. Based on that result, we expanded our study to evaluate the inhibitory effects of 22 monoterpenoids on CYP2B6 activity in vitro. Among the monoterpenoids screened, borneol, camphor, cineole, isoborneol, menthol, and perillaldehyde showed slight inhibition of CYP2B6-catalyzed bupropion hydroxylation, displaying greater than 50% inhibition at 50muM. Citral and geraniol strongly inhibited CYP2B6 hydroxylase activity in a competitive manner, with K(i) values of 6.8 and 10.3muM, respectively, which are higher than the K(i) (1.8muM) of the well-known CYP2B6-selective inhibitor thio-TEPA. These in vitro data indicate that high amounts of these two monoterpenoids might interact with drugs that are metabolized by CYP2B6. The in vivo pharmacokinetics of these compounds should be examined to determine whether the inhibition of CYP2B6 activity by monoterpenoids has clinical relevance.     

Pyruvate Formate-Lyase Interacts Directly with the Formate Channel FocA to Regulate Formate Translocation

The FNT (formate-nitrite transporters) form a superfamily of pentameric membrane channels that translocate monovalent anions across biological membranes. FocA (formate channel A) translocates formate bidirectionally but the mechanism underlying how translocation of formate is controlled and what governs substrate specificity remains unclear. Here we demonstrate that the normally soluble dimeric enzyme pyruvate formate-lyase (PflB), which is responsible for intracellular formate generation in enterobacteria and other microbes, interacts specifically with FocA. Association of PflB with the cytoplasmic membrane was shown to be FocA dependent and purified, Strep-tagged FocA specifically retrieved PflB from Escherichia coli crude extracts. Using a bacterial two-hybrid system, it could be shown that the N-terminus of FocA and the central domain of PflB were involved in the interaction. This finding was confirmed by chemical cross-linking experiments. Using constraints imposed by the amino acid residues identified in the cross-linking study, we provide for the first time a model for the FocA–PflB complex. The model suggests that the N-terminus of FocA is important for interaction with PflB. An in vivo assay developed to monitor changes in formate levels in the cytoplasm revealed the importance of the interaction with PflB for optimal translocation of formate by FocA. This system represents a paradigm for the control of activity of FNT channel proteins.     

Molecular Architecture of Photoreceptor Phosphodiesterase Elucidated by Chemical Cross-Linking and Integrative Modeling

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery and adaptation of visual detection. Although major steps in the PDE6 activation/deactivation pathway have been identified, mechanistic understanding of PDE6 regulation is limited by the lack of knowledge about the molecular organization of the PDE6 holoenzyme (αβγγ). Here, we characterize the PDE6 holoenzyme by integrative structural determination of the PDE6 catalytic dimer (αβ), based primarily on chemical cross-linking and mass spectrometric analysis. Our models built from high-density cross-linking data elucidate a parallel organization of the two catalytic subunits, with juxtaposed α-helical segments within the tandem regulatory GAF domains to provide multiple sites for dimerization. The two catalytic domains exist in an open configuration when compared to the structure of PDE2 in the apo state. Detailed structural elements for differential binding of the γ-subunit to the GAFa domains of the α- and β-subunits are revealed, providing insight into the regulation of the PDE6 activation/deactivation cycle.     

Modified nicotine metabolism in transgenic tobacco plants expressing the human cytochrome P450 2A6 cDNA

In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo.     

Analysis of peptides secreted from cultured mouse brain tissue

Peptides represent a major class of cell–cell signaling molecules. Most peptidomic studies have focused on peptides present in brain or other tissues. For a peptide to function in intercellular signaling, it must be secreted. The present study was undertaken to identify the major peptides secreted from mouse brain slices that were cultured in oxygenated buffer for 3–4 h. Approximately 75% of the peptides identified in extracts of cultured slices matched the previously reported peptide content of heat-inactivated mouse brain tissue, whereas only 2% matched the peptide content of unheated brain tissue; the latter showed a large number of postmortem changes. As found with extracts of heat-inactivated mouse brain, the extracts of cultured brain slices represented secretory pathway peptides as well as peptides derived from intracellular proteins such as those present in the cytosol and mitochondria. A subset of the peptides detected in the extracts of the cultured slices was detected in the culture media. The vast majority of secreted peptides arose from intracellular proteins and not secretory pathway proteins. The peptide RVD-hemopressin, a CB1 cannabinoid receptor agonist, was detected in culture media, which is consistent with a role for RVD-hemopressin as a non-classical neuropeptide. Taken together with previous studies, the present results show that short-term culture of mouse brain slices is an appropriate system to study peptide secretion, especially the non-conventional pathway(s) by which peptides produced from intracellular proteins are secreted. This article is part of a Special Issue entitled: An Updated Secretome.     

Analysis of the local dynamics of human insulin and a rapid-acting insulin analog by hydrogen/deuterium exchange mass spectrometry

Human insulin and insulin lispro (lispro), a rapid-acting insulin analog, have identical primary structures, except for the transposition of a pair of amino acids. This mutation results in alterations in their higher order structures, with lispro dissociating more easily than human insulin. In our previous study performed using hydrogen/deuterium exchange mass spectrometry (HDX/MS), differences were observed in the rates and levels of deuteration among insulin analog products, which were found to be related to their self-association stability. In this study, we carried out peptide mapping of deuterated human insulin and lispro to determine the regions responsible for these deuteration differences and to elucidate the type of structural changes that affect their HDX reactivity. We identified A3–6 and B22–24 as the 2 regions that showed distinct differences in the number of deuterium atoms incorporated between human insulin and lispro. These regions contain residues that are thought to participate in hexamerization and dimerization, respectively. We also determined that over time, the differences in deuteration levels decreased in A3–6, whereas they increased in B22–24, suggesting a difference in the dynamics between these 2 regions. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

A new derivative for oxosteroid analysis by mass spectrometry

Here we report a new method for oxosteroid identification utilizing “tandem mass tag hydrazine” (TMTH) carbonyl-reactive derivatisation reagent. TMTH is a reagent with a chargeable tertiary amino group attached through a linker to a carbonyl-reactive hydrazine group. Thirty oxosteroids were analysed after derivatisation with TMTH by electrospray ionization mass spectrometry (ESI-MS) and were found to give high ion-currents compared to underivatised molecules. ESI-tandem mass spectrometry (MS/MS) analysis of the derivatives yielded characteristic fragmentation patterns with specific mass reporter ions derived from the TMT group. A shotgun ESI-MS method incorporating TMTH derivatisation was applied to a urine sample.     

Comparison of molecular species of various transphosphatidylated phosphatidylserine (PS) with bovine cortex PS by mass spectrometry

The exogenous introduction of a molecular species mixture of bovine cortex phosphatidylserine (BC-PS) has been claimed to improve memory function in subjects suffering from age-associated memory impairment and dementia. However, it has been also reported that oral administration of another molecular species mixture of transphosphatidylated-soybean phosphatidylserine (T-Soy-PS) showed a little effect in older individuals with memory complaints. In this study, a new type of mixture of transphosphatidylated-fish liver phosphatidylserine (T-FL-PS) species, as well as intact molecular species of the two commercial products of T-Soy-PS made in the United States and Europe, were characterized by mass spectrometry and tandem mass spectrometry, and molecular species of various transphosphatidylated PSs, including T-FL-PS, T-Soy-PS and transphosphatidylated-squid skin phosphatidylserine (T-SS-PS) were then compared with those of BC-PS for the first time. The results show that (i) the presence of a relatively high content of docosahexaenoic acid (DHA)-containing species (more than 45%) is remarkable in T-FL-PS, (ii) DHA-ether PS species are found only in T-FL-PS, especially the species (about 17%) made from marine fish liver, rather than BC-PS and T-SS-PS, and (iii) DHA species present in both T-FL-PS and T-SS-PS are significantly enriched, compared with those in BC-PS (about 10%) and T-Soy-PS (no DHA species). We conclude that mixtures of T-FL-PS and T-SS-PS species are considered to be qualified alternatives of BC-PS supplement used as brain nutrients. It is expected that intact structural information on molecular species in current and potential transphosphatidylated PS products provided here will be useful in the further study and development of therapeutic roles of the phospholipid at molecular species level.     

Comparative analysis of secreted proteins from normal and preeclamptic trophoblastic cells using proteomic approaches

Preeclampsia (PE) is a pathology of pregnancy which represents the main cause of maternal and perinatal morbidity and mortality. Defective placentation is the first event of this pathology. The purpose of this study was to identify the proteins secreted by cytotrophoblastic cells (CTB) using proteomic approach that are associated with PE. Comparison of secreted proteins by mass spectrometry allowed us to identify 21 proteins which were significantly differentially secreted by control and PE CTB. One protein has been detected exclusively in supernatant of control CTB and was identified as factor XIII chain A. To determine if this observation is due to a difference of protein secretion or gene expression, its mRNA was quantified in all CTB. We found that it was significantly decreased in PE CTB compared to control. Collectively, these data suggest that decrease of factor XIII chain A might be associated with development of PE.     

Characterization of peptides resulting from digestion of human skin elastin with elastase

Several pathological disorders are associated with abnormalities in elastic fibers, which are mainly composed of elastin. Understanding the biochemical basis of such disorders requires information about the primary structure of elastin. Since the acquisition of structural information for elastin is hampered by its extreme insolubility in water or any organic solvent, in this study, human skin elastin was digested with elastase to produce water-soluble peptides. Tandem mass spectrometry (MS/MS) experiments were performed using conventional electrospray ionization (ESI) and nano-ESI techniques coupled with ion trap and quadrupole time-of-flight (qTOF) mass analyzers, respectively. The peptides were identified from the fragment spectra using database searching and/or de novo sequencing. The cleavage sites of the enzyme and, for the first time, the extent and location of proline hydroxylation in human skin elastin were determined. A total of 117 peptides were identified with sequence coverage of 58.8%. It has been observed that 25% of proline residues in the sequenced region are hydroxylated. Elastase cleaves predominantly at the C-terminals of the amino acids Gly, Val, Leu, Ala, and Ile, and to a lesser extent at Phe, Pro, Glu, and Arg. Our results confirm a previous report that human skin elastin lacks amino acid sequences expressed by exon 26A.     

A combined A431 cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening compounds from total alkaloid of Radix Caulophylli acting on the human EGFR

We have developed an online analytical method that combines A431 cell membrane chromatography (A431/CMC) with high performance liquid chromatography and mass spectrometry (LC/MS) for identifying active components from Radix Caulophylli acting on human EGFR. Retention fractions on A431/CMC model were captured onto an enrichment column and the components were directly analyzed by combining a 10-port column switcher with an LC/MS system for separation and preliminary identification. Using Sorafenib tosylate as a positive control, taspine and caulophine from Radix Caulophylli were identified as the active molecules which could act on the EGFR. This A431/CMC-online-LC/MS method can be applied for screening active components acting on EGFR from traditional Chinese medicines exemplified by Radix Caulophylli and will be of great utility in drug discovery using natural medicinal herbs as a source of novel compounds.     

Analysis of the human HP1 interactome reveals novel binding partners

Heterochromatin protein 1 (HP1) has first been described in Drosophila as an essential component of constitutive heterochromatin required for stable epigenetic gene silencing. Less is known about the three mammalian HP1 isotypes CBX1, CBX3 and CBX5. Here, we applied a tandem affinity purification approach coupled with tandem mass spectrometry methodologies in order to identify interacting partners of the mammalian HP1 isotypes. Our analysis identified with high confidence about 30–40 proteins co-eluted with CBX1 and CBX3, and around 10 with CBX5 including a number of novel HP1-binding partners. Our data also suggest that HP1 family members are mainly associated with a single partner or within small protein complexes composed of limited numbers of components. Finally, we showed that slight binding preferences might exist between HP1 family members.     

Comparative Multiplexed Mass Spectrometric Analyses of Endogenously Expressed Yeast Nuclear and Cytoplasmic Exosomes

Here we combined tandem affinity purification with several mass-spectrometry-based approaches to gain more insight into the composition and structure of the yeast nuclear-cytoplasmic exosome protein complex. The yeast exosome fulfills several different functions in RNA metabolism and can be localized in both the cytoplasm and the nucleus. These two exosome complexes differ in protein composition, although they share several constituents. We focused on these differences in composition by selecting a nuclear-specific exosome protein (Rrp6) and a cytoplasmic-specific protein (Ski7) as the tandem-affinity-purification-tagged affinity bait protein. First, we investigated both these purified exosome assemblies by macromolecular mass spectrometry (MS) to determine the stability and mass of the intact protein complexes and to obtain information on composition and core constituents. We used tandem MS on these intact protein complexes to further probe the composition and to obtain insight into the peripheral nature of some of the constituents. Finally, we combine stable isotope labeling with MS to quantitate differences in exosome composition and posttranslational modifications. We identified a few phosphorylation sites that are differentially regulated between the cytoplasmic exosome and the nuclear exosome. From all of these data, we conclude that the yeast nuclear exosome and the cytoplasmic exosome share a common stable core complex, but are decorated with quite a few differing peripheral proteins. We show that the nuclear exosome selectively copurifies with the α/β importin heterodimer, which is known to be involved in the transport of proteins across the nuclear membrane.     

Phosphorylation of Numb regulates its interaction with the clathrin-associated adaptor AP-2

Numb is thought to participate in clathrin-dependent endocytosis by directly interacting with the clathrin-associated adaptor complex AP-2, although the underlying mechanisms are unknown. Numb is also known to be phosphorylated at Ser(264)in vitro and in vivo. Here, we found that Numb is phosphorylated in vitro by Ca(2+)/calmodulin-dependent protein kinase I on Ser(283). This phosphorylation was also observed in transfected COS-7 cells, indicating its physiological relevance. Pull-down experiments showed that the phosphorylation of Numb impaired its binding to the AP-2 complex and simultaneously recruited 14-3-3 proteins in vitro. Based on experiments using Numb mutants, both the initial phosphorylation of Ser(264) and the subsequent phosphorylation of Ser(283) are sufficient to abolish the binding of Numb to AP-2 and to promote the interaction with 14-3-3 protein. These findings suggest a novel mechanism for the regulation of Numb-mediated endocytosis, namely through direct phosphorylation.     

Roles of superoxide and myeloperoxidase in ascorbate oxidation in stimulated neutrophils and H2O2-treated HL60 cells

Ascorbate is present at high concentrations in neutrophils and becomes oxidized when the cells are stimulated. We have investigated the mechanism of oxidation by studying cultured HL60 cells and isolated neutrophils. Addition of H₂O₂ to ascorbate-loaded HL60 cells resulted in substantial oxidation of intracellular ascorbate. Oxidation was myeloperoxidase-dependent, but not attributable to hypochlorous acid, and can be explained by myeloperoxidase (MPO) exhibiting direct ascorbate peroxidase activity. When neutrophils were stimulated with phorbol myristate acetate, about 40% of their intracellular ascorbate was oxidized over 20 min. Ascorbate loss required NADPH oxidase activity but in contrast to the HL60 cells did not involve myeloperoxidase. It did not occur when exogenous H₂O₂ was added, was not inhibited by myeloperoxidase inhibitors, and was the same for normal and myeloperoxidase-deficient cells. Neutrophil ascorbate loss was enhanced when endogenous superoxide dismutase was inhibited by cyanide or diethyldithiocarbamate and appears to be due to oxidation by superoxide. We propose that in HL60 cells, MPO-dependent ascorbate oxidation occurs because cellular ascorbate can access newly synthesized MPO before it becomes packaged in granules: a mechanism not possible in neutrophils. In neutrophils, we estimate that ascorbate is capable of competing with superoxide dismutase for a small fraction of the superoxide they generate and propose that the superoxide responsible is likely to come from previously identified sites of intracellular NADPH oxidase activity. We speculate that ascorbate might protect the neutrophil against intracellular effects of superoxide generated at these sites.     

Development and validation of a rapid and sensitive assay for simultaneous quantification of midazolam, 1′-hydroxymidazolam,and 4-hydroxymidazolam by liquid chromatography coupled to tandem mass-spectrometry

Midazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity. A rapid, sensitive, and selective LC–MS/MS method was developed for simultaneous quantitation of midazolam and its metabolites (1′-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D₅) analog of midazolam was utilized as an internal standard. Sample preparation either from human plasma (100 μL) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 μL) with an average recovery of >90% for all compounds. The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2.1 mm × 100 mm, 3.5 μm) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B). The flow rate was 0.25 mL/min and total run time was 5.5 min. Calibration curves were linear over the concentration range of 0.100–250 ng/mL. The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes. The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85–115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites. This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam.     

Tryptophan 334 oxidation in bovine cytochrome c oxidase subunit I involves free radical migration

A single tryptophan (W(334(I))) within the mitochondrial-encoded core subunits of cytochrome c oxidase (CcO) is selectively oxidized when hydrogen peroxide reacts with the binuclear center. W(334(I)) is converted to hydroxytryptophan as identified by reversed-phase HPLC-electrospray ionization tandem mass spectrometry analysis of peptides derived from the three SDS-PAGE purified subunits. Total sequence coverage of subunits I, II and III was limited to 84%, 66% and 54%, respectively. W(334(I)) is located on the surface of CcO at the membrane interface. Two other surface tryptophans within nuclear-encoded subunits, W(48(IV)) and W(19(VIIc)), are also oxidized when hydrogen peroxide reacts with the binuclear center (Musatov et al. (2004) Biochemistry 43, 1003-1009). Two aromatic-rich networks of amino acids were identified that link the binuclear center to the three oxidized tryptophans. We propose the following mechanism to explain these results. Electron transfer through the aromatic networks moves the free radicals generated at the binuclear center to the surface-exposed tryptophans, where they produce hydroxytryptophan.     

An efficient and convenient synthesis of deuterium-labelled seminolipid isotopomers and their ESI-MS characterization

Seminolipids 1a and 1b and galactosylalkylacylglycerols 2a and 2b, labelled with deuterium on the alkyl or acyl chain, respectively, were obtained isotopically and chemically pure through a straightforward synthesis from protected glycidyl galactoside 3 in an overall 22% yield. The identity and purity of compounds was ascertained by NMR spectroscopy and ESI mass spectrometry analysis. These labelled compounds are important as internal standards for quantification of these lipids by mass spectrometry, and they could also be used in metabolic studies in in vitro and even in vivo systems. Extension of the procedure could provide a route for the preparation of isotopomers of other compounds of the same general class.