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.     

Good Manufacturing Practices (GMP) manufacturing of advanced therapy medicinal products: a novel tailored model for optimizing performance and estimating costs

Background aimsAdvanced therapy medicinal products (ATMP) have gained considerable attention in academia due to their therapeutic potential. Good Manufacturing Practice (GMP) principles ensure the quality and sterility of manufacturing these products. We developed a model for estimating the manufacturing costs of cell therapy products and optimizing the performance of academic GMP-facilities.MethodsThe “Clean-Room Technology Assessment Technique” (CTAT) was tested prospectively in the GMP facility of BCRT, Berlin, Germany, then retrospectively in the GMP facility of the University of California-Davis, California, USA. CTAT is a two-level model: level one identifies operational (core) processes and measures their fixed costs; level two identifies production (supporting) processes and measures their variable costs. The model comprises several tools to measure and optimize performance of these processes. Manufacturing costs were itemized using adjusted micro-costing system.ResultsCTAT identified GMP activities with strong correlation to the manufacturing process of cell-based products. Building best practice standards allowed for performance improvement and elimination of human errors. The model also demonstrated the unidirectional dependencies that may exist among the core GMP activities. When compared to traditional business models, the CTAT assessment resulted in a more accurate allocation of annual expenses. The estimated expenses were used to set a fee structure for both GMP facilities. A mathematical equation was also developed to provide the final product cost.ConclusionsCTAT can be a useful tool in estimating accurate costs for the ATMPs manufactured in an optimized GMP process. These estimates are useful when analyzing the cost-effectiveness of these novel interventions.     

Oligomers Containing 2′-Deoxyinosine Isosteres as Ambiguous Nucleoside or 1,3-Propanediol as Nucleoside Substitute

Abstract

Three new appropriately protected phosphoramidites have been synthesized. Two of them (1 and 2) are isosteric to that of inosine (3) [1], one is a derivative of 1′3-propanediol (4). Whereas the inosine isosteres contain an ambiguous base recognizing adenine, guanine as well as cytosine residues in double stranded DNA-fragments the 1,3-propanediol unit can be seen as a simple nucleoside substitute in a DNA chain. It contains only those structural elements necessary to form the sugar/phosphate backbone, without supplying the DNA with either a base [21 or a 2′-deoxyribofuranosyl moiety.     

Menthol as an alternative anaesthetic and sedative for rainbow trout,Oncorhynchus mykiss

Menthol is known for its analgesic properties, but relatively little information is available on its potential as an anaesthetic on fish. The purpose of this study was to assess anaesthetic and sedative effectiveness of menthol and its safety in rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout 180 ± 28 g (mean ± SD) within a range of 152–208 g fish^?1 were individually exposed to menthol concentrations between 10 and 150 mg l^?1 and observed for behavioural responses, induction time to anaesthesia and recovery time. Menthol concentrations of 40–150 mg l^?1 induced anaesthesia with varying exposure times. There was an exponential relationship (p < 0.001) between induction time and menthol concentration. Menthol concentrations of 80–150 mg l^?1 induced anaesthesia within three minutes of exposure and fish recovered within three minutes. Induction and recovery data showed that 80 mg l^?1 was most suitable for anaesthesia in juveniles of this species. Concentrations of 10–20 mg l^?1 had sedative effects. Menthol stock solutions prepared using ethanol and acetone and storage time of stock solutions at room temperature for up to 48 h showed no significant differences in anaesthetic efficiency. When exposure time to menthol was kept constant at three minutes, 22% of fish had temporary cessation of gill ventilation. These fish had longer recovery times than those that did not show that response. Menthol was an effective anaesthetic and could be tested as a sedative for trout.     

Soil carbon and nitrogen dynamics along a latitudinal transect in Western Siberia, Russia

An 1800-km South to North transect (N 53°43′ to 69°43′) through Western Siberia was established to study the interaction of nitrogen and carbon cycles. The transect comprised all major vegetation zones from steppe, through taiga to tundra and corresponded to a natural temperature gradient of 9.5°C mean annual temperature (MAT). In order to elucidate changes in the control of C and N cycling along this transect, we analyzed physical and chemical properties of soils and microbial structure and activity in the organic and in the mineral horizons, respectively. The impact of vegetation and climate exerted major controls on soil C and N pools (e.g., soil organic matter, total C and dissolved inorganic nitrogen) and process rates (gross N mineralization and heterotrophic respiration) in the organic horizons. In the mineral horizons, however, the impact of climate and vegetation was less pronounced. Gross N mineralization rates decreased in the organic horizons from south to north, while remaining nearly constant in the mineral horizons. Especially, in the northern taiga and southern tundra gross nitrogen mineralization rates were higher in the mineral compared to organic horizons, pointing to strong N limitation in these biomes. Heterotrophic respiration rates did not exhibit a clear trend along the transect, but were generally higher in the organic horizon compared to mineral horizons. Therefore, C and N mineralization were spatially decoupled at the northern taiga and tundra. The climate change implications of these findings (specifically for the Arctic) are discussed.     

Indicators of the Ecological Impact of Bottom-Trawl Disturbance on Seabed Communities

The Ecosystem Approach to Fisheries requires that managers take account of the environmental impacts of fishing. We develop linked state and pressure indicators that show the impact of bottom-trawling on benthic communities. The state indicator measures the proportion of an area where benthic invertebrate biomass (B) or production (P) is more than 90% of pristine benthic biomass (B _0.9) or production (P_0.9). The pressure indicator measures the proportion of the area where trawling frequency is sufficiently high to prevent reaching predicted B_0.9 or P_0.9. Time to recovery to B_0.9 and P_0.9 after trawling, depending on the habitat, was estimated using a validated size-based model of the benthic community. Based on trawling intensity in 2003, 53.5% of the southern North Sea was trawled too frequently for biomass to reach B_0.9, and 27.1% was trawled too frequently for production to reach P_0.9. As a result of bottom-trawling in 2003, in 56% of the southern North Sea benthic biomass was below B_0.9, whereas in 27% of the southern North Sea benthic production was below P_0.9. Modeled recovery times were comparable to literature estimates (2.5 to more than 6 years). The advantages of using the area with an ecological impact of trawling as a pressure indicator are that it is conceptually easy to understand, it responds quickly to changes in management action, it can be implemented at a relevant scale for fisheries management, and the necessary effort distribution data are centrally collected. One of this approach’s greatest utilities, therefore, will be to communicate to policy makers and fishing enterprises the expected medium- to long-term ecological benefits that will accrue if the frequency of trawling in particular parts of fishing grounds is reduced.     

A decision support tool for integrated fisheries bycatch management

Reviews in Fish Biology and Fisheries - Participatory decision tools enable stakeholders to reconcile conflicting natural resources management objectives. Fisheries targeting highly productive...     

早期液体复苏对感染性休克患者血流动力学的影响

目的：早期液体复苏对感染性休克患者血流动力学的影响。方法：选取2012年2月-2013年2月我院ICU收治的26例感染性休克患者作为研究对象,随机分为对照组和试验组,各13例。两组患者均采用PICCO监测,并根据早期复苏目标导向（Earlygoaldirectedtherapy,EGDT）进行早期液体复苏治疗。对照组和试验组复苏液分别为林格液和6％羟乙基淀粉130／0．4氯化钠溶液。分别于复苏开始时（Oh）、8h和24h收集患者的血流动力学参数。结果：两组患者CO及PAWP水平均随着时间的延长下降,而CI、CVP及SVR水平均随着时间的增加上升。除对照组CI外,与开始复苏（oh）相比较试验组和对照组的C0、CI、CVP、SVR及PAWP与开始复苏（O小时）相比较均有显著差异（P值均〈0．05）。经重复测量资料的．方差分析进行比较发现,与对照组相比较,试验组CVP和SVR上升水平及PAWP下降水平明显,差异具有统计学意义（P值均〈0．05）。结论：感染性休克患者使用6％羟乙基淀粉130／0．4氯化钠溶液进行复苏,能更好的改善患者的血流动力学指标。     

INPPO Actions and Recognition as a Driving Force for Progress in Plant Proteomics: Change of Guard,INPPO Update,and Upcoming Activities

The International Plant Proteomics Organization (INPPO) is a non‐profit organization whose members are scientists involved or interested in plant proteomics. Since the publication of the first INPPO highlights in 2012, continued progress on many of the organization's mandates/goals has been achieved. Two major events are emphasized in this second INPPO highlights. First, the change of guard at the top, passing of the baton from Dominique Job, INPPO founding President to Ganesh Kumar Agrawal as the incoming President. Ganesh K. Agrawal, along with Dominique Job and Randeep Rakwal initiated the INPPO. Second, the most recent INPPO achievements and future targets, mainly the organization of first the INPPO World Congress in 2014, tentatively planned for Hamburg (Germany), are mentioned.     

Structures of Atg7-Atg3 and Atg7-Atg10 reveal noncanonical mechanisms of E2 recruitment by the autophagy E1

Central to most forms of autophagy are two ubiquitin-like proteins (UBLs), Atg8 and Atg12, which play important roles in autophagosome biogenesis, substrate recruitment to autophagosomes, and other aspects of autophagy. Typically, UBLs are activated by an E1 enzyme that (1) catalyzes adenylation of the UBL C terminus, (2) transiently covalently captures the UBL through a reactive thioester bond between the E1 active site cysteine and the UBL C terminus, and (3) promotes transfer of the UBL C terminus to the catalytic cysteine of an E2 conjugating enzyme. The E2, and often an E3 ligase enzyme, catalyzes attachment of the UBL C terminus to a primary amine group on a substrate. Here, we summarize our recent work reporting the structural and mechanistic basis for E1-E2 protein interactions in autophagy.