Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7

The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography–MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs.Malaria is a global health problem with 214 million new cases of malaria and 438,000 deaths reported in 2015, mostly in sub-Saharan Africa (1). The endoperoxide class of antimalarial drugs, such as artemisinin (ART), is the first line of defense against malaria infection against a backdrop of multidrug-resistant parasites (2) and lack of effective vaccines (3, 4). Given the effectiveness of the ART class, the question arises: how do these drugs kill parasites? A suggested mechanism of action involves the cleavage of the endoperoxide bridge by a source of Fe²⁺ or heme. This cleavage results in the formation of oxyradicals that rearrange into primary or secondary carbon-centered radicals. These radicals have been proposed to alkylate parasite proteins that somehow result in the death of the parasite (5). However, this proposal remains a subject of intense debate (6, 7), while these alkylated proteins are yet to be formally identified. So far, the proposed targets of ART action include a PfATP6 enzyme, the Plasmodium falciparum ortholog of mammalian sarcoendoplasmic reticulum Ca²¹-ATPases (SERCAs) (5), translational controlled tumor protein, and heme (5). Additionally, Haynes et al. (8) proposed that ART may act by impairing parasite redox homeostasis as a consequence of an interaction between the drug and flavin adenine dinucleotide (FADH) and/or other parasite flavoenzymes in the parasite, leading to the generation of reactive oxygen species (ROS). New approaches are required for definitive identification of ART molecular targets. This insight into the drug activation-dependent mechanism of action will be invaluable in the target-led development of more potent drugs with the potential to circumvent the emergence of resistance to current first-line ART-based therapies. The goal of this study was to identify ART-targeted proteins and their interacting partners in P. falciparum. We recently adopted a proteomic approach developed by Speers and Cravatt (9) to synthesize a suite of pyrethroid activity-based protein profiling probes (ABPPs) (10). Using alkyne/azide-coupling partners through “click chemistry,” we identified several cytochrome P450 enzymes that metabolized deltamethrin in rat liver microsomes (10). More recently, a chemical proteomic approach was developed to identify parasite proteins targeted by an albitiazolium antimalarial drug candidate in situ using a photoactivation cross-linking approach (11). However, this generic approach can introduce significant promiscuity in the proteins tagged based on the intracompartmental distribution of drug independent of actual mechanisms.Here, we introduced the design and synthesis of click chemistry-compatible activity-based probes incorporating the endoperoxide scaffold of ART as a warhead to alkylate and identified the ART molecular target(s) in asexual stages of the malaria parasite (Fig. 1). A major advantage of this strategy is that the reporter tags are introduced under “click” reaction conditions performed after the drug has achieved its biological effects, enabling purification, identification, and quantification of alkylated parasite’s proteins and their interacting partners as shown in Fig. 1B. To avoid nonspecific probe-dependent tagging, a common limitation of these approaches, we generated the respective “control” nonperoxide partners to improve the specificity and biological relevance of our resultant tagged protein list.Open in a separate windowFig. 1.Rational design of the ART-ABPPs. (A) Conversion of ART to ART-ABPPs involves the addition of a clickable handle (i.e., an alkyne or azide to the ART drug pharmacophore by the peptide-coupling method illustrated in SI Text). The structures of the alkyne (P1) and azide (P2) probes and respective inactive deoxy controls CP1 and CP2 with in vitro IC₅₀ values are presented. (B) General workflow of copper-catalyzed and copper-free click chemistry approaches used in the identification of alkylated proteins after in situ treatment of P. falciparum parasite with alkyne and azide ART-ABPPs. The azide- and alkyne-modified proteins are tagged with biotin azide and biotin dibenzocyclooctyne (Biotin-DIBO), respectively, via click reactions followed by affinity purification tandem with LC-MS/MS for protein identification.     

Lithium,Inositol and Mitochondria

Our recent DNA-microarray and proteomics studies searching for pathways affected both by chronic lithium treatment and by knockout of each of two genes (IMPA1 or Slc5a3) encoding for proteins related to inositol metabolism, indicated up-regulation of mitochondria-related genes and autophagy-related proteins in the frontal cortex. Differently from previously reported observations of aberrant mitochondrial function in bipolar patients which leave a causality relationship between mitochondrial dysfunction and bipolar disorder an open question, the behavioral results of our recent report following rotenone treatment tempt us to speculate that mitochondrial dysfunction predisposes manic behavior and that drugs targeted to ameliorate mitochondrial function are potential preventers of bursting manic episodes. However, the promiscuity of the involvement of mitochondrial dysfunction and impaired autophagy in the pathophysiology of psychiatric and neurodegenerative disorders raises questions regarding the credibility and relevance of these findings.     

Left ventricular diastolic function in relation to the urinary proteome: A proof-of-concept study in a general population

Background

In previous studies, we identified two urinary proteomic classifiers, termed HF1 and HF2, which discriminated subclinical diastolic left ventricular (LV) dysfunction from normal. HF1 and HF2 combine information from 85 and 671 urinary peptides, mainly up- or down-regulated collagen fragments. We sought to validate these classifiers in a population study.

Methods

In 745 people randomly recruited from a Flemish population (49.8 years; 51.3% women), we measured early and late diastolic peak velocities of mitral inflow (E and A) and mitral annular velocities (e' and a') by conventional and tissue Doppler echocardiography, and the urinary proteome by capillary electrophoresis coupled with mass spectrometry.

Results

In the analyses adjusted for sex, age, body mass index, blood pressure, heart rate, LV mass index and intake of medications, we expressed effect sizes per 1-SD increment in the classifiers. HF1 was associated with 0.204 cm/s lower e' peak velocity (95% confidence interval, 0.057–0.351; p = 0.007) and 0.145 higher E/e' ratio (0.023–0.268; p = 0.020), while HF2 was associated with a 0.174 higher E/e' ratio (0.046–0.302; p = 0.008). According to published definitions, 67 (9.0%) participants had impaired LV relaxation and 96 (12.9%) had elevated LV filling pressure. The odds of impaired relaxation associated with HF1 was 1.38 (1.01–1.88; p = 0.043) and that of increased LV filling pressure associated with HF2 was 1.38 (1.00–1.90; p = 0.052).

Conclusions

In a general population, the urinary proteome correlated with diastolic LV dysfunction, proving its utility for early diagnosis of this condition.     

Mapping the diatom redox-sensitive proteome provides insight into response to nitrogen stress in the marine environment

Diatoms are ubiquitous marine photosynthetic eukaryotes responsible for approximately 20% of global photosynthesis. Little is known about the redox-based mechanisms that mediate diatom sensing and acclimation to environmental stress. Here we used a quantitative mass spectrometry-based approach to elucidate the redox-sensitive signaling network (redoxome) mediating the response of diatoms to oxidative stress. We quantified the degree of oxidation of 3,845 cysteines in the Phaeodactylum tricornutum proteome and identified approximately 300 redox-sensitive proteins. Intriguingly, we found redox-sensitive thiols in numerous enzymes composing the nitrogen assimilation pathway and the recently discovered diatom urea cycle. In agreement with this finding, the flux from nitrate into glutamine and glutamate, measured by the incorporation of ¹⁵N, was strongly inhibited under oxidative stress conditions. Furthermore, by targeting the redox-sensitive GFP sensor to various subcellular localizations, we mapped organelle-specific oxidation patterns in response to variations in nitrogen quota and quality. We propose that redox regulation of nitrogen metabolism allows rapid metabolic plasticity to ensure cellular homeostasis, and thus is essential for the ecological success of diatoms in the marine ecosystem.Aerobic organisms produce reactive oxygen species (ROS) as a byproduct of oxygen-based metabolic pathways, such as photosynthesis, photorespiration, and oxidative phosphorylation (1). Perturbations in oxygenic metabolism under various stress conditions can induce oxidative stress from overproduction of ROS (2, 3). Because ROS are highly reactive forms of oxygenic metabolites, critical mechanisms for ROS detoxification have evolved consisting of ROS-scavenging enzymes and small molecules, including glutathione (GSH) (4). As the most abundant low molecular weight thiol antioxidant, GSH has critical roles in maintaining a proper cellular thiol–disulfide balance and in detoxifying H₂O₂ via the ascorbate–GSH cycle (5).Although classically ROS were considered toxic metabolic byproducts that ultimately lead to cell death, it is now recognized that ROS act as central secondary messengers involved in compartmentalized signaling networks (1, 6–8). Modulation of various cell processes by ROS signaling is mediated largely by posttranslational thiol oxidation, whereby their physical structure and biochemical activity are modified upon oxidation (9). Thus, the redox states of these proteins possess crucial information needed for cell acclimation to stress conditions (10, 11). The emergence of advanced redox proteomic approaches, such as the OxICAT method (12), has created new opportunities to identify redox-sensitive proteins (e.g., redoxome) on the system level and to quantify their precise level of oxidation on exposure to environmental stress conditions.Marine photosynthetic microorganisms (phytoplankton) are the basis of marine food webs. Despite the fact that their biomass represents only approximately 0.2% of the photosynthetic biomass on earth, they are responsible for nearly 50% of the annual global carbon-based photosynthesis and greatly influence the global biogeochemical carbon cycle (13). This high ratio of productivity to biomass, reflected in high turnover rates, makes phytoplankton highly responsive to climate change. Phytoplankton can grow rapidly and form massive blooms that stretch over hundreds of kilometers in the oceans and are regulated by such environmental factors as nutrient availability and biotic interactions with grazers and viruses.Diatoms are a highly diverse clade of phytoplankton, responsible for roughly 20% of global primary productivity (14). Consequently, diatoms play a central role in the biogeochemical cycling of important nutrients, including carbon, nitrogen, and silica, which constitute part of their ornate cell wall. As members of the eukaryotic group known as stramenopiles (or heterokonts), diatoms are derived from a secondary endosymbiotic event involving red and green algae engulfed within an ancestral protest (15).The unique multilineage content of diatom genomes reveals a melting pot of biochemical characteristics that resemble bacterial, plant, and animal traits, including the integration of a complete urea cycle, fatty acid oxidation in the mitochondria, and plant C4-like related pathways (16, 17). During bloom succession, phytoplankton cells are subjected to diverse environmental stress conditions that lead to ROS production, such as allelopathic interactions (18), CO₂ availability (19, 20), UV exposure (21), iron limitation (22), and viral infection (23). Recently reported evidence suggests that diatoms possess a surveillance system based on the induction of ROS that have been implicated in response to various environmental stresses (22, 24). Nevertheless, very little is known about cell signaling processes in marine phytoplankton and their potential role in acclimation to rapid fluctuations in the chemophysical gradients in the marine environment (25).Using a mass spectrometry-based approach, we examined the diatom redoxome and quantified its degree of oxidation under oxidative stress conditions. The wealth of recently identified redox-sensitive proteins participating in various cellular functions suggests a fundamental role of redox regulation in diatom biology. We mapped the redox-sensitive enzymes into a metabolic network and evaluated their role in the adjustment of metabolic flux under variable environmental conditions. We further explored the redox sensitivity of the primary nitrogen-assimilating pathway and demonstrated the role of compartmentalized redox regulation in cells under nitrogen stress conditions using a redox-sensitive GFP sensor targeted to specific subcellular localizations.     

Identification and characterization of the constituent human serum antibodies elicited by vaccination

Most vaccines confer protection via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT⁺ serum IgG repertoire comprises ∼100 antibody clonotypes, with three clonotypes accounting for >40% of the response. All 13 recombinant IgGs examined bound to vaccine antigen with K_d ∼ 10⁻⁸–10⁻¹⁰ M. Five of 13 IgGs recognized the same linear epitope on TT, occluding the binding site used by the toxin for cell entry, suggesting a possible explanation for the mechanism of protection conferred by the vaccine. Importantly, only a small fraction (<5%) of peripheral blood plasmablast clonotypes (CD3⁻CD14⁻CD19⁺CD27⁺⁺CD38⁺⁺CD20⁻TT⁺) at the peak of the response (day 7), and an even smaller fraction of memory B cells, were found to encode antibodies that could be detected in the serological memory response 9 mo postvaccination. This suggests that only a small fraction of responding peripheral B cells give rise to the bone marrow long-lived plasma cells responsible for the production of biologically relevant amounts of vaccine-specific antibodies (near or above the K_d). Collectively, our results reveal the nature and dynamics of the serological response to vaccination with direct implications for vaccine design and evaluation.Most approved vaccines confer protection against infectious diseases by the induction of long-lived plasma cells (LLPCs), which secrete antibodies that serve to neutralize and opsonize the pathogen for many years or decades (1–3). Additionally, the generation of memory B cells (mBCs) provides both a mechanism for the rapid synthesis of affinity matured, antigen-specific antibodies following rechallenge and a means to diversify the humoral immune response to confer protection against rapidly evolving viruses or bacteria (4). Although some vaccines elicit antibody titers that remain virtually constant for many decades, for others, including the tetanus toxoid (TT) vaccine, antibody titers wane monotonically over time (5). Booster immunization triggers the rapid expansion and differentiation of cognate B cells, generating antigen-specific plasmablasts that peak in concentration in peripheral blood after 6–7 d and subsequently rapidly decline to nearly undetectable levels (6, 7). Some, but not all, of these peak-wave plasmablasts migrate to specialized niches overwhelmingly located in the bone marrow (BM) and survive as LLPCs (8), which constitute the major source of all classes of Ig in the serum (9).The establishment of serological memory following either primary or booster vaccination is not understood well (10–14). Even though antibody production is the most critical effector function of B-cell immunity, and antigen-specific antibodies in the serum play a key role in protection against pathogen challenge, technical difficulties have precluded direct determination of the identities of the mAbs that comprise the serum antibody response to vaccination. However, recent studies showing that flu vaccination elicits not only neutralizing antibodies but also antibodies that enhance infection by different flu strains (15) underscore the pressing need to develop approaches for delineating the sequences and functionalities of the serum antibodies elicited by vaccination (16).Single-cell cloning has been used to identify neutralizing antibodies encoded by mBCs or plasmablasts in peripheral blood (17). However, although extremely useful for understanding of the structural mechanisms that can lead to the blockade of pathogen infection, the interrogation of single peripheral B cells alone cannot provide information on whether antibodies encoded by single B cells are also produced as secreted IgGs from BM LLPCs, and hence whether they contribute to the serological memory induced by vaccination. A detailed understanding of the diversity of serum antibodies elicited by vaccination, their functionality (e.g., antigen affinity, epitope specificity), and their relative concentrations in the blood can provide key insights toward vaccine evaluation and development.Here, we deployed high-resolution liquid chromatography (LC) tandem MS (MS/MS) (18–20) for the molecular-level analysis of the serum IgG repertoire, combined with deep sequencing of the V gene repertoire of peripheral B lymphocyte subsets (20) and subsequent expression and characterization of representative serum antibodies, to map the dynamics of the human humoral response to vaccination in unprecedented detail. We elected to analyze the response to booster immunization of the TT vaccine because (i) it elicits a highly effective neutralizing response that is protective toward Clostridium tetani challenge; (ii) the vaccine is highly efficacious, and as a result, no deaths from tetanus intoxication have been reported in the United States for individuals who have completed at least primary immunization (21); (iii) TT has been used as a model for analyzing B-cell development following vaccination in humans (6, 22, 23); and (iv) although early serological and mAb studies had pointed to the C-terminal fragment of the toxin heavy chain [recombinant TT fragment C (rTT.C)] as the target for antibody-mediated protection (24), the precise mechanism by which antibodies elicited by the vaccine mediate neutralization has remained unclear.We show that the anti-TT serum IgG repertoire at steady state is composed of a limited number of antibody clonotypes (∼80–100) displaying uniformly high antigen affinity (low nanomolar or subnanomolar), that most of the serum repertoire postboost comprises preexisting (i.e., prevaccination) serum antibody clonotypes, and that there is only partial overlap between the peak-wave plasmablast V gene repertoire and the TT⁺ serum IgG repertoire at steady state after vaccination. We identified several serum monoclonal IgGs that bind to rTT.C, and epitope mapping revealed that all rTT.C-specific antibodies tested bind to an immunodominant linear epitope at the ganglioside-binding site of the toxin that is used for cell entry. Computational antibody docking substantiated that binding of these antibodies to the toxin blocks access to the ganglioside ligand, thus providing a possible mechanistic explanation for how the TT vaccine confers protection. These results highlight the importance of understanding the composition and dynamics of the serum antibody repertoire, together with the V gene repertoire in peripheral B lymphocytes, for the molecular understanding of vaccine function.     

A proteomic evaluation of the effects of the pharmaceuticals diclofenac and gemfibrozil on marine mussels (Mytilus spp.): evidence for chronic sublethal effects on stress‐response proteins

Human pharmaceuticals (e.g. the lipid regulator gemfibrozil and the non‐steroidal anti‐inflammatory drug diclofenac) are an emerging environmental threat in the aquatic environment. This study aimed to evaluate sublethal effects of these two commonly found pharmaceuticals on the protein profiles of marine mussels (Mytilus spp.). Mytilus spp. was exposed to environmentally relevant and elevated concentrations (1 and 1000 µg/l respectively) of both drugs for 14 days. In addition, mussels were maintained for seven days post treatment to examine the potential of blue mussels to recover from such an exposure. Differential protein expression signatures (PES) in the digestive gland of mussels were obtained using two‐dimensional gel electrophoresis after 7, 14, and 21 days of exposure. Twelve spots were significantly increased or decreased by gemfibrozil and/or diclofenac, seven of which were successfully identified by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis. These proteins were involved in energy metabolism, oxidative stress response, protein folding, and immune responses. Changes in the PES over time suggested that mussels were still experiencing oxidative stress for up to seven days post exposure. In addition, a suite of biomarkers comprising glutathione transferase, lipid peroxidation, and DNA damage were studied. An oxidative stress response was confirmed by biomarker responses. To our knowledge, this is the first investigation using proteomics to assess the potential effects of human pharmaceuticals on a non‐target species in an environmentally‐relevant model. The successful application of this proteomic approach supports its potential use in pollution biomonitoring and highlights its ability to aid in the discovery of new biomarkers. Copyright © 2013 John Wiley & Sons, Ltd.     

一种基于卷积神经网络的DIA数据预处理模型

目的数据非依赖性采集(data independent acquisition,DIA)是目前针对大通量蛋白质组学分析常用的一种数据采集方式。在对DIA数据无目标的分析方式中,由于无法预测肽段出现在DIA数据中的位置,需要对谱中所有的峰进行分析。但谱中含有大量的噪声峰,这些峰会严重影响后续蛋白质定性定量分析的效率与效果,所以在DIA数据的无目标分析过程中先进行预处理以去除噪声峰就成了很重要的一步。为了能充分利用从DIA数据中提取出来的肽段在一级质谱(first stage of mass spectrometry,MS1)和二级质谱(second stage of mass spectrometry,MS2)中的峰信息,提出质谱卷积神经网络(mass spectrometry convolutional neural network,MSCNN)模型。方法不同于传统的方法,本文首先提出适用于MSCNN网络结构的样本提取流程,然后利用MSCNN对样本进行训练和学习,该模型可以最大限度利用肽在MS1和MS2中的特征,最后通过观察模型在测试集中的结果来验证模型的效果。结果和传统算法相比,在保证真峰处理效果大致相同的情况下,MSCNN模型过滤噪声峰的数量提高了约11.2%。结论本文提出的MSCNN模型可以更有效地去除DIA数据中的噪声峰。