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.     

Ordinary ethics: lay people's deliberations on social sex selection

Abstract

This article summarises the results of a research project that used a scenario about sex selection of embryos for social reasons as a basis for discussion groups with lay people. The aim of the research was to examine the processes by which non-professionals make ethical evaluations in relation to a contested area in medical genetics. We note in particular the role played in the discussions by expressions of instinct; making distinctions; rational argument; reference to principles; use of personal experience; analogies and examples; slippery slope arguments and meta-reflections. The implications for developing processes of public consultation and debate are also considered.     

A Vulnerability Assessment of 300 Species in Florida: Threats from Sea Level Rise,Land Use,and Climate Change

Species face many threats, including accelerated climate change, sea level rise, and conversion and degradation of habitat from human land uses. Vulnerability assessments and prioritization protocols have been proposed to assess these threats, often in combination with information such as species rarity; ecological, evolutionary or economic value; and likelihood of success. Nevertheless, few vulnerability assessments or prioritization protocols simultaneously account for multiple threats or conservation values. We applied a novel vulnerability assessment tool, the Standardized Index of Vulnerability and Value, to assess the conservation priority of 300 species of plants and animals in Florida given projections of climate change, human land-use patterns, and sea level rise by the year 2100. We account for multiple sources of uncertainty and prioritize species under five different systems of value, ranging from a primary emphasis on vulnerability to threats to an emphasis on metrics of conservation value such as phylogenetic distinctiveness. Our results reveal remarkable consistency in the prioritization of species across different conservation value systems. Species of high priority include the Miami blue butterfly (Cyclargus thomasi bethunebakeri), Key tree cactus (Pilosocereus robinii), Florida duskywing butterfly (Ephyriades brunnea floridensis), and Key deer (Odocoileus virginianus clavium). We also identify sources of uncertainty and the types of life history information consistently missing across taxonomic groups. This study characterizes the vulnerabilities to major threats of a broad swath of Florida’s biodiversity and provides a system for prioritizing conservation efforts that is quantitative, flexible, and free from hidden value judgments.     

Aspergillus fumigatus Invasion Increases with Progressive Airway Ischemia

Despite the prevalence of Aspergillus-related disease in immune suppressed lung transplant patients, little is known of the host-pathogen interaction. Because of the mould’s angiotropic nature and because of its capacity to thrive in hypoxic conditions, we hypothesized that the degree of Aspergillus invasion would increase with progressive rejection-mediated ischemia of the allograft. To study this relationship, we utilized a novel orthotopic tracheal transplant model of Aspergillus infection, in which it was possible to assess the effects of tissue hypoxia and ischemia on airway infectivity. Laser Doppler flowmetry and FITC-lectin were used to determine blood perfusion, and a fiber optic microsensor was used to measure airway tissue oxygen tension. Fungal burden and depth of invasion were graded using histopathology. We demonstrated a high efficacy (80%) for producing a localized fungal tracheal infection with the majority of infection occurring at the donor-recipient anastomosis; Aspergillus was more invasive in allogeneic compared to syngeneic groups. During the study period, the overall kinetics of both non-infected and infected allografts was similar, demonstrating a progressive loss of perfusion and oxygenation, which reached a nadir by days 10-12 post-transplantation. The extent of Aspergillus invasion directly correlated with the degree of graft hypoxia and ischemia. Compared to the midtrachea, the donor-recipient anastomotic site exhibited lower perfusion and more invasive disease; a finding consistent with clinical experience. For the first time, we identify ischemia as a putative risk factor for Aspergillus invasion. Therapeutic approaches focused on preserving vascular health may play an important role in limiting Aspergillus infections.     

Structural and Physiological Analyses of the Alkanesulphonate-Binding Protein (SsuA) of the Citrus Pathogen Xanthomonas citri

Background

The uptake of sulphur-containing compounds plays a pivotal role in the physiology of bacteria that live in aerobic soils where organosulfur compounds such as sulphonates and sulphate esters represent more than 95% of the available sulphur. Until now, no information has been available on the uptake of sulphonates by bacterial plant pathogens, particularly those of the Xanthomonas genus, which encompasses several pathogenic species. In the present study, we characterised the alkanesulphonate uptake system (Ssu) of Xanthomonas axonopodis pv. citri 306 strain (X. citri), the etiological agent of citrus canker.

Methodology/Principal Findings

A single operon-like gene cluster (ssuEDACB) that encodes both the sulphur uptake system and enzymes involved in desulphurisation was detected in the genomes of X. citri and of the closely related species. We characterised X. citri SsuA protein, a periplasmic alkanesulphonate-binding protein that, together with SsuC and SsuB, defines the alkanesulphonate uptake system. The crystal structure of SsuA bound to MOPS, MES and HEPES, which is herein described for the first time, provides evidence for the importance of a conserved dipole in sulphate group coordination, identifies specific amino acids interacting with the sulphate group and shows the presence of a rather large binding pocket that explains the rather wide range of molecules recognised by the protein. Isolation of an isogenic ssuA-knockout derivative of the X. citri 306 strain showed that disruption of alkanesulphonate uptake affects both xanthan gum production and generation of canker lesions in sweet orange leaves.

Conclusions/Significance

The present study unravels unique structural and functional features of the X. citri SsuA protein and provides the first experimental evidence that an ABC uptake system affects the virulence of this phytopathogen.