Aspergillus-specific antibodies – Targets and applications

Aspergillus is a fungal genus comprising several hundred species, many of which can damage the health of plants, animals and humans by direct infection and/or due to the production of toxic secondary metabolites known as mycotoxins. Aspergillus-specific antibodies have been generated against polypeptides, polysaccharides and secondary metabolites found in the cell wall or secretions, and these can be used to detect and monitor infections or to quantify mycotoxin contamination in food and feed. However, most Aspergillus-specific antibodies are generated against heterogeneous antigen preparations and the specific target remains unknown. Target identification is important because this can help to characterize fungal morphology, confirm host penetration by opportunistic pathogens, detect specific disease-related biomarkers, identify new candidate targets for antifungal drug design, and qualify antibodies for diagnostic and therapeutic applications. In this review, we discuss how antibodies are raised against heterogeneous Aspergillus antigen preparations and how they can be characterized, focusing on strategies to identify their specific antigens and epitopes. We also discuss the therapeutic, diagnostic and biotechnological applications of Aspergillus-specific antibodies.     

β-Arrestins as Potential Therapeutic Targets for Alzheimer’s Disease

β-arrestins represent a small family of G protein-coupled receptors (GPCRs) regulators, which provide modulating effects by facilitating desensitization and internalization of GPCRs as well as initiating their own signalings. Recent reports have demonstrated that β-arrestins levels were correlated with amyloid-β peptide (Aβ) pathology in brains of Alzheimer’s disease (AD) patients and animal models. β-arrestins could enhance the activity of γ-secretase via interacting with anterior pharynx defective 1 subunit, which increased Aβ production and contributed to the pathogenesis of AD. In addition, Aβ-induced internalization of β2-adrenergic receptor internalization and loss of dendritic spine in neurons were proven to be mediated by β-arrestins, further establishing their pathogenic role in AD. More importantly, deletion of β-arrestins markedly attenuated AD pathology, without causing any gross abnormality. Here, we review the evidence about the roles of β-arrestins in the progression of AD. In addition, the established and postulated mechanisms by which β-arrestins mediated in AD pathogenesis are also discussed. Based on the role of β-arrestins in AD pathogenesis, genetically or pharmacologically targeting β-arrestins might provide new opportunities for AD treatment.     

Targets for AD treatment: conflicting messages from γ-secretase inhibitors

Current evidence suggests that Alzheimer's disease (AD) is a multi-factorial disease that starts with accumulation of multiple proteins. We have previously proposed that inhibition of γ-secretase may impair membrane recycling causing neurodegeneration starting at synapses (Sambamurti K., Suram A., Venugopal C., Prakasam A., Zhou Y., Lahiri D. K. and Greig N. H. A partial failure of membrane protein turnover may cause Alzheimer's disease: a new hypothesis. Curr. Alzheimer Res., 3, 2006, 81). We also proposed familal AD mutations increase Aβ42 by inhibiting γ-secretase. Herein, we discuss the failure of Eli Lilly's γ-secretase inhibitor, semagacestat, in clinical trials in the light of our hypothesis, which extends the problem beyond toxicity of Aβ aggregates. We elaborate that γ-secretase inhibitors lead to accumulation of amyloid precursor protein C-terminal fragments that can later be processed by γ-secretase to yields bursts of Aβ to facilitate aggregation. Although we do not exclude a role for toxic Aβ aggregates, inhibition of γ-secretase can affect numerous substrates other than amyloid precursor protein to affect multiple pathways and the combined accumulation of multiple peptides in the membrane may impair its function and turnover. Taken together, protein processing and turnover pathways play an important role in maintaining cellular homeostasis and unless we clearly see consistent disease-related increase in their levels or activity, we need to focus on preserving their function rather than inhibiting them for treatment of AD and similar diseases.     

T Cell Receptor Signaling Pathways： New Targets for Herpes Simplex Virus

Herpes simplex viruses (HSV-1 and HSV-2) cause global morbidity and synergistically correlate with HIV infection.HSV exists life-long in a latent form in sensory neurons with intermittent reactivation,in despite of host immune surveillance.While abundant evidence for HSV interfering with innate immune responses so as to favor the replication and propagation of the virus,several lines of evidence declare that HSV attenuates adaptive immunity by various mechanisms,including but not limited to the ablation of antigen presentation,induction of apoptosis,and interruption of cellular signaling.In this review,we will focus on the perturbative role of HSV in Tcells signaling.     

Quercetin Targets Cysteine String Protein (CSPα) and Impairs Synaptic Transmission

Background

Cysteine string protein (CSPα) is a synaptic vesicle protein that displays unique anti-neurodegenerative properties. CSPα is a member of the conserved J protein family, also called the Hsp40 (heat shock protein of 40 kDa) protein family, whose importance in protein folding has been recognized for many years. Deletion of the CSPα in mice results in knockout mice that are normal for the first 2–3 weeks of life followed by an unexplained presynaptic neurodegeneration and premature death. How CSPα prevents neurodegeneration is currently not known. As a neuroprotective synaptic vesicle protein, CSPα represents a promising therapeutic target for the prevention of neurodegenerative disorders.

Methodology/Principal Findings

Here, we demonstrate that the flavonoid quercetin promotes formation of stable CSPα-CSPα dimers and that quercetin-induced dimerization is dependent on the unique cysteine string region. Furthermore, in primary cultures of Lymnaea neurons, quercetin induction of CSPα dimers correlates with an inhibition of synapse formation and synaptic transmission suggesting that quercetin interfers with CSPα function. Quercetin''s action on CSPα is concentration dependent and does not promote dimerization of other synaptic proteins or other J protein family members and reduces the assembly of CSPα:Hsc70 units (70kDa heat shock cognate protein).

Conclusions/Significance

Quercetin is a plant derived flavonoid and popular nutritional supplement proposed to prevent memory loss and altitude sickness among other ailments, although its precise mechanism(s) of action has been unclear. In view of the therapeutic promise of upregulation of CSPα and the undesired consequences of CSPα dysfunction, our data establish an essential proof of principle that pharmaceutical agents can selectively target the neuroprotective J protein CSPα.     

Arachidonic Acid-metabolizing Cytochrome P450 Enzymes Are Targets of ω-3 Fatty Acids

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against cardiovascular disease by largely unknown mechanisms. We tested the hypothesis that EPA and DHA may compete with arachidonic acid (AA) for the conversion by cytochrome P450 (CYP) enzymes, resulting in the formation of alternative, physiologically active, metabolites. Renal and hepatic microsomes, as well as various CYP isoforms, displayed equal or elevated activities when metabolizing EPA or DHA instead of AA. CYP2C/2J isoforms converting AA to epoxyeicosatrienoic acids (EETs) preferentially epoxidized the ω-3 double bond and thereby produced 17,18-epoxyeicosatetraenoic (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) from EPA and DHA. We found that these ω-3 epoxides are highly active as antiarrhythmic agents, suppressing the Ca²⁺-induced increased rate of spontaneous beating of neonatal rat cardiomyocytes, at low nanomolar concentrations. CYP4A/4F isoforms ω-hydroxylating AA were less regioselective toward EPA and DHA, catalyzing predominantly ω- and ω minus 1 hydroxylation. Rats given dietary EPA/DHA supplementation exhibited substantial replacement of AA by EPA and DHA in membrane phospholipids in plasma, heart, kidney, liver, lung, and pancreas, with less pronounced changes in the brain. The changes in fatty acids were accompanied by concomitant changes in endogenous CYP metabolite profiles (e.g. altering the EET/EEQ/EDP ratio from 87:0:13 to 27:18:55 in the heart). These results demonstrate that CYP enzymes efficiently convert EPA and DHA to novel epoxy and hydroxy metabolites that could mediate some of the beneficial cardiovascular effects of dietary ω-3 fatty acids.     

WNT/β-Catenin Signaling Regulates Multiple Steps of Myogenesis by Regulating Step-Specific Targets

Molecules involved in WNT/β-catenin signaling show specific spatiotemporal expression and play vital roles in myogenesis; however, it is still largely unknown how WNT/β-catenin signaling regulates each step of myogenesis. Here, we show that WNT/β-catenin signaling can control diverse biological processes of myogenesis by regulating step-specific molecules. In order to identify the temporally specific roles of WNT/β-catenin signaling molecules in muscle development and homeostasis, we used in vitro culture systems for both primary mouse myoblasts and C2C12 cells, which can differentiate into myofibers. We found that a blockade of WNT/β-catenin signaling in the proliferating cells decreases proliferation activity, but does not induce cell death, through the regulation of genes cyclin A2 (Ccna2) and cell division cycle 25C (Cdc25c). During muscle differentiation, the inhibition of WNT/β-catenin signaling blocks myoblast fusion through the inhibition of the Fermitin family homolog 2 (Fermt2) gene. Blocking WNT/β-catenin signaling in the well-differentiated myofibers results in the failure of maintenance of their structure by disruption of cadherin/β-catenin/actin complex formation, which plays a crucial role in connecting a myofiber''s cytoskeleton to the surrounding extracellular matrix. Thus, our results indicate that WNT/β-catenin signaling can regulate multiple steps of myogenesis, including cell proliferation, myoblast fusion, and homeostasis, by targeting step-specific molecules.     

Substrate Trapping Proteomics Reveals Targets of the βTrCP2/FBXW11 Ubiquitin Ligase

Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study βTrCP2/FBXW11, a substrate adaptor for the SKP1–CUL1–F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between FBXW11 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to “trap” ubiquitylated substrates on the SCF^FBXW11 E3 complex. Comparative mass spectrometry analysis of immunopurified FBXW11 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. In focused studies, we found that SCF^FBXW11 bound, polyubiquitylated, and destabilized RAPGEF2, a guanine nucleotide exchange factor that activates the small GTPase RAP1. High RAPGEF2 protein levels promoted cell-cell fusion and, consequently, multinucleation. Surprisingly, this occurred independently of the guanine nucleotide exchange factor (GEF) catalytic activity and of the presence of RAP1. Our data establish new functions for RAPGEF2 that may contribute to aneuploidy in cancer. More broadly, this report supports the continued use of substrate trapping proteomics to comprehensively define targets for E3 ubiquitin ligases. All proteomic data are available via ProteomeXchange with identifier PXD001062.     

Extracellular M. tuberculosis DNA Targets Bacteria for Autophagy by Activating the Host DNA-Sensing Pathway

Polarity in mammalian cells emerges from the assembly of signaling molecules into extensive biochemical interaction networks. Despite their complexity, bacterial pathogens have evolved parsimonious mechanisms to hijack these systems. Here, we develop a tractable experimental and theoretical model to uncover fundamental operating principles, in both mammalian cell polarity and bacterial pathogenesis. Using synthetic derivatives of the enteropathogenic Escherichia coli guanine-nucleotide exchange factor (GEF) Map, we discover that Cdc42 GTPase signal transduction is controlled by the interaction between Map and F-actin. Mathematical modeling reveals how actin dynamics coupled to a Map-dependent positive feedback loop spontaneously polarizes Cdc42 on the plasma membrane. By rewiring the pathogenic signaling circuit to operate through β-integrin stimulation, we further show how Cdc42 is polarized in response to an extracellular spatial cue. Thus, a molecular pathway of polarity is proposed, centered on the interaction between GEFs and F-actin, which is likely to function in diverse biological systems.     

Impact of ‘Stretch’ Targets for Cardiovascular Disease Management within a Local Pay-for-Performance Programme

Pay-for-performance programs are often aimed to improve the management of chronic diseases. We evaluate the impact of a local pay for performance programme (QOF+), which rewarded financially more ambitious quality targets (‘stretch targets’) than those used nationally in the Quality and Outcomes Framework (QOF). We focus on targets for intermediate outcomes in patients with cardiovascular disease and diabetes. A difference-in-difference approach is used to compare practice level achievements before and after the introduction of the local pay for performance program. In addition, we analysed patient-level data on exception reporting and intermediate outcomes utilizing an interrupted time series analysis. The local pay for performance program led to significantly higher target achievements (hypertension: p-value <0.001, coronary heart disease: p-values <0.001, diabetes: p-values <0.061, stroke: p-values <0.003). However, the increase was driven by higher rates of exception reporting (hypertension: p-value <0.001, coronary heart disease: p-values <0.03, diabetes: p-values <0.05) in patients with all conditions except for stroke. Exception reporting allows practitioners to exclude patients from target calculations if certain criteria are met, e.g. informed dissent of the patient for treatment. There were no statistically significant improvements in mean blood pressure, cholesterol or HbA1c levels. Thus, achievement of higher payment thresholds in the local pay for performance scheme was mainly attributed to increased exception reporting by practices with no discernable improvements in overall clinical quality. Hence, active monitoring of exception reporting should be considered when setting more ambitious quality targets. More generally, the study suggests a trade-off between additional incentive for better care and monitoring costs.     

New Aspects of an Old Drug – Diclofenac Targets MYC and Glucose Metabolism in Tumor Cells

Non-steroidal anti-inflammatory drugs such as diclofenac exhibit potent anticancer effects. Up to now these effects were mainly attributed to its classical role as COX-inhibitor. Here we show novel COX-independent effects of diclofenac. Diclofenac significantly diminished MYC expression and modulated glucose metabolism resulting in impaired melanoma, leukemia, and carcinoma cell line proliferation in vitro and reduced melanoma growth in vivo. In contrast, the non-selective COX inhibitor aspirin and the COX-2 specific inhibitor NS-398 had no effect on MYC expression and glucose metabolism. Diclofenac significantly decreased glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 1 (MCT1) gene expression in line with a decrease in glucose uptake and lactate secretion. A significant intracellular accumulation of lactate by diclofenac preceded the observed effect on gene expression, suggesting a direct inhibitory effect of diclofenac on lactate efflux. While intracellular lactate accumulation impairs cellular proliferation and gene expression, it does not inhibit MYC expression as evidenced by the lack of MYC regulation by the MCT inhibitor α-cyano-4-hydroxycinnamic acid. Finally, in a cell line with a tetracycline-regulated c-MYC gene, diclofenac decreased proliferation both in the presence and absence of c-MYC. Thus, diclofenac targets tumor cell proliferation via two mechanisms, that is inhibition of MYC and lactate transport. Based on these results, diclofenac holds potential as a clinically applicable MYC and glycolysis inhibitor supporting established tumor therapies.     

HNF4α Combinatorial Isoform Heterodimers Activate Distinct Gene Targets that Differ from Their Corresponding Homodimers

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Modular Organization of α-Toxins from Scorpion Venom Mirrors Domain Structure of Their Targets,Sodium Channels

To gain success in the evolutionary “arms race,” venomous animals such as scorpions produce diverse neurotoxins selected to hit targets in the nervous system of prey. Scorpion α-toxins affect insect and/or mammalian voltage-gated sodium channels (Na_vs) and thereby modify the excitability of muscle and nerve cells. Although more than 100 α-toxins are known and a number of them have been studied into detail, the molecular mechanism of their interaction with Na_vs is still poorly understood. Here, we employ extensive molecular dynamics simulations and spatial mapping of hydrophobic/hydrophilic properties distributed over the molecular surface of α-toxins. It is revealed that despite the small size and relatively rigid structure, these toxins possess modular organization from structural, functional, and evolutionary perspectives. The more conserved and rigid “core module” is supplemented with the “specificity module” (SM) that is comparatively flexible and variable and determines the taxon (mammal versus insect) specificity of α-toxin activity. We further show that SMs in mammal toxins are more flexible and hydrophilic than in insect toxins. Concomitant sequence-based analysis of the extracellular loops of Na_vs suggests that α-toxins recognize the channels using both modules. We propose that the core module binds to the voltage-sensing domain IV, whereas the more versatile SM interacts with the pore domain in repeat I of Na_vs. These findings corroborate and expand the hypothesis on different functional epitopes of toxins that has been reported previously. In effect, we propose that the modular structure in toxins evolved to match the domain architecture of Na_vs.     

Thiamin (Vitamin B1) Biosynthesis and Regulation: A Rich Source of Antimicrobial Drug Targets?

Drug resistance of pathogens has necessitated the identification of novel targets for antibiotics. Thiamin (vitamin B1) is an essential cofactor for all organisms in its active form thiamin diphosphate (ThDP). Therefore, its metabolic pathways might be one largely untapped source of antibiotics targets. This review describes bacterial thiamin biosynthetic, salvage, and transport pathways. Essential thiamin synthetic enzymes such as Dxs and ThiE are proposed as promising drug targets. The regulation mechanism of thiamin biosynthesis by ThDP riboswitch is also discussed. As drug targets of existing antimicrobial compound pyrithiamin, the ThDP riboswitch might serves as alternative targets for more antibiotics.