Mechanism of pH-independent Cleavage and Isomerization of 3′-Ribonucleotide Phosphodiesters

Abstract

The detailed mechanisms of pH-independent cleavage and isomerization reactions of the phosphodiester bond are evaluated by examining kinetics of hydrolytic reactions of selected mono- and dialkyl esters of 3′-uridylic acid. The mechanistic choices are made by comparing LFER's between reaction rates and acidity of the alkyl group of phosphodi- and triesters.     

Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes

Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)¹ probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188.ATP binding and hydrolysis are the driving processes in all living organisms. Hundreds of cellular proteins are able to bind and hydrolyze ATP to unfold proteins, transport molecules over membranes, or phosphorylate small molecules or proteins. Proteins with very different structures are able to bind ATP. A large and important class of ATP binding proteins is that of the kinases, which transfer the gamma phosphate from ATP to substrates. Kinases, and particularly protein kinases, play pivotal roles in signaling and protein regulation.The genome of the model plant Arabidopsis thaliana encodes for over 1099 protein kinases and hundreds of other ATP binding proteins (1, 2). Protein kinases are involved in nearly all signaling cascades and regulate processes ranging from cell cycle to flowering and from immunity to germination. Many protein kinases in plants are receptor-like kinases (RLKs), often carrying extracellular leucine-rich repeats (LRRs). The RLK class contains at least 610 members (3), including famous examples such as receptors involved in development (e.g. BRI1, ER, CLV1) and immunity (e.g. FLS2, EFR). Other important classes are mitogen-activated protein (MAP) kinases (MPKs) (20 different members), MPK kinase kinase kinases (MAP3Ks) (60 different members (4)), and calcium-dependent protein kinases (CPKs) (34 different members (5)). Because of their diverse and important roles, protein kinases have been intensively studied in plant science. The current approach is to study protein kinases individually—a daunting task, considering the remaining hundreds of uncharacterized protein kinases. New approaches are necessary in order to study protein kinases and other ATP binding proteins globally rather than individually.ATP binding activities of protein kinases and other proteins can be detected globally by acyl-ATP (AcATP) probes (6, 7) (Fig. 1A). AcATP binds to the ATP pocket of ATP binding proteins and places the acyl group in close proximity to conserved lysine residues in the ATP binding pocket. The acyl phosphonate moiety serves as an electrophilic warhead that can be nucleophilically attacked by the amino group of the lysine, resulting in a covalent attachment of the acyl reporter of the AcATP probe on the lysine and a concomitant release of ATP. The reporter tag is usually a biotin to capture and identify the labeled proteins. Labeled proteins can be displayed on protein blots using streptavidin-HRP. However, because AcATP labels many ATP binding proteins and protein kinases are of relatively low abundance, mass spectrometry is more often used to identify and quantify labeling with AcATP probes. The analysis is preferably done using Xsite, a procedure that involves trypsination of the entire labeled proteome, followed by analysis of the biotinylated peptides rather than the biotinylated proteins (8). This “KiNativ ” approach provides enough depth and resolving power to monitor ∼160 protein kinases in a crude mammalian proteome (7). Of the 518 human protein kinases (9), 394 (76%) have been detected via AcATP labeling (6).Open in a separate windowFig. 1.Structure and mechanism of labeling with BHAcATP. A, BHAcATP contains ATP, an acyl phosphate reactive group, and a biotin tag. When BHAcATP binds to the ATP binding pocket of a protein, the amino group of the nearby lysine reacts with the carbonyl carbon, which results in the covalent binding of the biotin tag to the protein while ATP is released. B, typical BHAcATP labeling profile of Arabidopsis leaf proteome. Arabidopsis leaf extracts were labeled with BHAcATP and the biotinylated proteins were detected on protein blots using streptavidin-HRP. Coomassie Brilliant Blue staining indicates equal loading. Asterisks indicate endogenously biotinylated proteins MCCA and BCCP. White, black, and gray arrowheads indicate bands containing ATBP+RBCL, PGK1, and a mix of ATP binding proteins, respectively. Abbreviations: MCCA, 3-methylcrotonyl-CoA carboxylase; BCCP, biotin carboxyl carrier protein; ATPB, chloroplastic ATPase; RBCL, ribulose-bisphosphate carboxylase; PGK1, phosphoglycerate kinase-1.KiNativ has mostly been used to validate targets of human drugs that target protein kinases using competitive labeling experiments. This approach has been used to identify selective inhibitors of, for example, Parkinson''s disease protein kinase LRRK2 (10), the BMK1 and JNK MAP kinases (11, 12), and the mTOR kinase (13). Importantly, the correlation of the biological activity of protein-kinase-inhibiting drugs with inhibitor affinity detected using KiNativ is better than that achieved when affinities are determined by assays using heterologously expressed protein kinases (7). This improved correlation illustrates that assays in the native environment provide a more realistic measure of protein kinase function.In addition to characterizing inhibitors selectively, AcATP probes can also display differential ATP binding activities of protein kinases. For example, labeling with AcATP probes during infection with dengue virus displayed a 2- to 8-fold activation of a DNA-dependent protein kinase (14) Similarly, AcATP labeling revealed an unexpected Raf kinase activation in extracts upon protein kinase inhibitor treatment (7). In conclusion, profiling with AcATP probes is a powerful approach for monitoring protein kinases and offers unprecedented opportunities to identify selective protein kinase inhibitors and discover protein kinases with differential ATP binding activities.In this work, we introduce AcATP profiling of plant proteomes. In addition to the analysis of labeled peptides, we characterized labeling using gel-based approaches and discovered that biotin is often oxidized in this procedure. We also performed an in-depth analysis of labeling sites in proteins other than protein kinases, which had not been done before. We discuss labeling outside known nucleotide binding pockets and investigate the correlation of labeling sites with protein abundance. We describe 63 labeling sites of known nucleotide binding pockets, of which 24 represent a remarkable diversity of protein kinases, including several LRR-RLKs. This work launches a new approach to study ATP binding proteins in plant science.     

Background levels of hydrogen cyanide in human breath measured by infrared cavity ring down spectroscopy

Hydrogen cyanide (HCN) in breath has been suggested as a diagnostic tool for cyanide poisoning and for cyanide-producing bacterial infections. To distinguish elevated levels of breath HCN, baseline data are needed. Background levels of HCN were measured in mixed exhaled air from 40 healthy subjects (26 men, 14 women, age 21–61 years; detection limit: 1.5?ppb; median: 4.4?ppb; range <1.5–14?ppb) by near-infrared cavity ring down spectroscopy (CRDS). No correlation was observed with smoking habits, recent meals or age. However, female subjects had slightly higher breath levels of HCN than male subjects. CRDS has not previously been used for this purpose.     

Extending the scope of the controlled logical clock

Event traces are helpful in understanding the performance behavior of parallel applications since they allow the in-depth analysis of communication and synchronization patterns. However, the absence of synchronized clocks on most cluster systems may render the analysis ineffective because inaccurate relative event timings may misrepresent the logical event order and lead to errors when quantifying the impact of certain behaviors or confuse the users of time-line visualization tools by showing messages flowing backward in time. In our earlier work, we have developed a scalable algorithm called the controlled logical clock that eliminates inconsistent inter-process timings postmortem in traces of pure MPI applications, potentially running on large processor configurations. In this paper, we first demonstrate that our algorithm also proves beneficial in computational grids, where a single application is executed using the combined computational power of several geographically dispersed clusters. Second, we present an extended version of the algorithm that—in addition to message-passing event semantics—also preserves and restores shared-memory event semantics, enabling the correction of traces from hybrid applications.     

Increased Summer Temperatures Reduce the Growth and Regeneration of Larix sibirica in Southern Boreal Forests of Eastern Kazakhstan

The larch forests at the southern limit of the Siberian boreal forest in Central Asia have repeatedly experienced strong recent growth declines attributed to decreasing summer precipitation in the course of climate warming. Here, we present evidence from the southernmost Larix sibirica forests in eastern Kazakhstan that these declines are primarily caused by a decrease in effective moisture due to increasing summer temperatures, despite constant annual, and summer precipitation. Tree-ring chronologies (>800 trees) showed a reduction by 50–80% in mean ring width and an increase in the frequency of missing rings since the 1970s. Climate-response analysis revealed a stronger (negative) effect of summer temperature (in particular of the previous year’s June and July temperature) on radial growth than summer precipitation (positive effect). It is assumed that a rise in the atmospheric vapor pressure deficit, which typically increases with temperature, is negatively affecting tree water status and radial growth, either directly or indirectly through reduced soil moisture. Larch rejuvenation ceased in the 1950s, which is partly explained by increasing topsoil desiccation in a warmer climate and a high drought susceptibility of larch germination, as was demonstrated by a germination experiment with variable soil moisture levels. The lack of regeneration and the reduced annual stem increment suggest that sustainable forest management aiming at timber harvesting is no longer feasible in these southern boreal forests. Progressive climate warming is likely to cause a future northward shift of the southern limit of the boreal forest.     

Anammox bacterial populations in deep marine hypersaline gradient systems

To extend the knowledge of anaerobic ammonium oxidation (anammox) habitats, bacterial communities were examined in two hypersaline sulphidic basins in Eastern Mediterranean Sea. The 2 m thick seawater–brine haloclines of the deep anoxic hypersaline basins Bannock and L’Atalante were sampled in intervals of 10 cm with increasing salinity. ¹⁵N isotope pairing incubation experiments showed the production of ²⁹N₂ and ³⁰N₂ gases in the chemoclines, ranging from 6.0 to 9.2 % salinity of the L’Atalante basin. Potential anammox rates ranged from 2.52 to 49.65 nmol N₂ L^?1 day^?1 while denitrification was a major N₂ production pathway, accounting for more than 85.5 % of total N₂ production. Anammox-related 16S rRNA genes were detected along the L’Atalante and Bannock haloclines up to 24 % salinity, and the amplification of the hydrazine synthase genes (hzsA) further confirmed the presence of anammox bacteria in Bannock. Fluorescence in situ hybridisation and sequence analysis of 16S rRNA genes identified representatives of the marine anammox genus ‘Candidatus Scalindua’ and putatively new operational taxonomic units closely affiliated to sequences retrieved in marine environments that have documented anammox activity. ‘Scalindua brodae’ like sequences constituted up to 84.4 % of the sequences retrieved from Bannock. The anammox community in L’Atalante was different than in Bannock and was stratified according to salinity increase. This study putatively extends anammox bacterial habitats to extremely saline sulphidic ecosystems.     

Ecological characteristics associated with high mobility in night-active moths

Mobility is an important factor influencing the range and persistence of local populations. However, mobility is very difficult to measure empirically and thus is poorly known in most taxa. Since ecological characteristics have been suggested as good estimators of mobility, we here explore the association between ecological characteristics and mobility. We surveyed night-active moths on a Swedish island, situated 16 km from the mainland, and compared ecological characteristics of the non-resident moths found on the island with those of a species pool of assumed potential vagrants from the neighbouring mainland. Species associated with high mobility were characterised by a large range, a high population density, an activity period during warm temperatures and by being habitat generalists or preferring open habitats. The generally assumed view of poly- and oligophagous species being more mobile than monophagous species was obscured by the effect of population density. Poly- and oligophagous species had higher population densities than did monophagous species, which probably explain their higher mobility found in this study. Our result highlights the need to consider the influence of ecological characteristics on mobility. This in turn will have implications for an increased understanding of distribution patterns, population persistence and how to prioritise conservation actions, especially since habitats and climate are under dramatic changes. In taxa where data on mobility are poor, ecological characteristics can be used as a proxy for mobility.