Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants

A wide-ranging examination of plastid (pt)DNA sequence homologies within higher plant nuclear genomes (promiscuous DNA) was undertaken. Digestion with methylation-sensitive restriction enzymes and Southern analysis was used to distinguish plastid and nuclear DNA in order to assess the extent of variability of promiscuous sequences within and between plant species. Some species, such as Gossypium hirsutum (cotton), Nicotiana tabacum (tobacco), and Chenopodium quinoa, showed homogenity of these sequences, while intraspecific sequence variation was observed among different cultivars of Pisum sativum (pea), Hordeum vulgare (barley), and Triticum aestivum (wheat). Hypervariability of plastid sequence homologies was identified in the nuclear genomes of Spinacea oleracea (spinach) and Beta vulgaris (beet), in which individual plants were shown to possess a unique spectrum of nuclear sequences with ptDNA homology. This hypervariability apparently extended to somatic variation in B. vulgaris. No sequences with ptDNA homology were identified by this method in the nuclear genome of Arabidopsis thaliana.      

Matrix Metalloproteinase 10 Degradomics in Keratinocytes and Epidermal Tissue Identifies Bioactive Substrates With Pleiotropic Functions*

Matrix metalloproteinases (MMPs) are important players in skin homeostasis, wound repair, and in the pathogenesis of skin cancer. It is now well established that most of their functions are related to processing of bioactive proteins rather than components of the extracellular matrix (ECM). MMP10 is highly expressed in keratinocytes at the wound edge and at the invasive front of tumors, but hardly any non-ECM substrates have been identified and its function in tissue repair and carcinogenesis is unclear. To better understand the role of MMP10 in the epidermis, we employed multiplexed iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS) and monitored MMP10-dependent proteolysis over time in secretomes from keratinocytes. Time-resolved abundance clustering of neo-N termini classified MMP10-dependent cleavage events by efficiency and refined the MMP10 cleavage site specificity by revealing a so far unknown preference for glutamate in the P1 position. Moreover, we identified and validated the integrin alpha 6 subunit, cysteine-rich angiogenic inducer 61 and dermokine as novel direct MMP10 substrates and provide evidence for MMP10-dependent but indirect processing of phosphatidylethanolamine-binding protein 1. Finally, we sampled the epidermal proteome and degradome in unprecedented depth and confirmed MMP10-dependent processing of dermokine in vivo by TAILS analysis of epidermis from transgenic mice that overexpress a constitutively active mutant of MMP10 in basal keratinocytes. The newly identified substrates are involved in cell adhesion, migration, proliferation, and/or differentiation, indicating a contribution of MMP10 to local modulation of these processes during wound healing and cancer development. Data are available via ProteomeXchange with identifier PXD002474.Matrix metalloproteinases (MMPs)¹ are extracellular zinc-dependent endoproteinases that are highly expressed in tissues undergoing remodeling processes during development, in response to injury, or as a result of neoplastic transformation (1–3). MMP10, also known as stromelysin-2, gained particular interest in the skin, because of its specific and strong expression in wound edge keratinocytes as well as at the invasive front of epithelial tumors (4–6). Overexpression of a constitutively active MMP10 mutant in wound keratinocytes in mice led to scattering of these cells at the tip of the migrating wound epithelium, altered β1-integrin expression, reduced AKT phosphorylation and increased apoptosis (7). Lack of MMP10 in a lung infection model affected genes that are involved in the regulation of apoptosis, cell proliferation, immune response and signal transduction (8). In the gut, bone marrow-derived MMP10 had a protective role in experimental colitis with implications in macrophage polarization (9). MMP10 released from hepatocytes and macrophages positively contributed to liver regeneration (10), whereby it promoted hepatocarcinogenesis in a complicated crosstalk with chemokine signaling (11). Most recently, Rohani et al. demonstrated a role for macrophage-derived MMP10 in moderating scar formation by controlling collagenase activity of dermal macrophages (12).Similar complex phenotypes have been associated with activities of most MMPs that, however, were not related to processing of extracellular matrix (ECM) proteins, the classical MMP substrates (13), but of bioactive mediators, including cell surface receptors, growth factor binding proteins, proteases, inhibitors, cytokines, and chemokines (14, 15). This changed the view on MMPs as simple tissue degraders to precise modulators of diverse processes, such as cell proliferation, migration, differentiation, angiogenesis, apoptosis and immune response (2, 16). As an example, functions of MMP3, the closest homolog of MMP10, in keratinocyte differentiation (17), tumor cell invasion (18), and immune cell recruitment (19) could be explained by processing of non-ECM proteins that have been identified as direct substrates of this protease (2, 20) in addition to ECM components (21). However, because MMP10 has been mostly neglected in the quest for new MMP substrates, it remains to be elucidated, if it also exerts its functions in part by processing of bioactive proteins whose identification is instrumental in understanding the mechanisms of action of MMP10 in tissue repair and carcinogenesis.Recently, we applied iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS), a multiplexed quantitative proteomics workflow for identification of protease substrates in complex proteomes (22–24), to reveal new targets of MMP10 in secretomes from mouse embryonic fibroblasts (25). Moreover, to mimic MMP10 activity at the epidermal–dermal interface, we devised a new workflow that allowed monitoring both cellular origins and cleavages of substrates in mixed secretomes from keratinocytes and fibroblasts (26). However, this study focused on basement membrane components and missed additional information on cleavage kinetics. Thus, in this work, we employed time-resolved TAILS to identify novel MMP10 substrates in keratinocyte secretomes and mouse epidermal tissue, aiming at further characterizing the MMP10 substrate degradome in epidermal keratinocytes for a better understanding of its biological roles in the skin. Here, we identified novel bioactive substrates of MMP 10 in vitro and in the skin in vivo, which provide insight into its functions in wound repair and carcinogenesis. In addition, we revealed an unexpected preference of MMP10 for substrates that harbor a glutamate residue in P1 position, which might be exploited for the development of specific activity-based probes or inhibitors for this important wound- and tumor-related protease.     

Thermal unfolding of soybean peroxidase. Appropriate high denaturant concentrations induce cooperativity allowing the correct measurement of thermodynamic parameters

We have earlier reported that both guanidine hydrochloride (GdnHCl)-induced and heat-induced unfolding of seed coat soybean peroxidase (SBP), monitored by far UV CD, show single step transition. However, although GdnHCl-induced unfolding follows a two-state pathway, the heat-induced denaturation proceeds through intermediates as indicated by the very low cooperativity of transition. In the former case, analysis of the data based on the two-state model gives true thermodynamic parameters, whereas underestimated values are obtained in the latter case. Available complex equations also cannot be applied for the analysis of the thermal unfolding of SBP due to the absence of separate transitions for the intermediates. In the present study, we report a method to obtain true thermodynamic parameters from thermal transition curves of SBP using the two-state model. When SBP is subjected to thermal unfolding at high GdnHCl concentrations (5.8-6.9 M), cooperative behavior is observed, which allowed the analysis by the two-state model to determine their thermodynamic parameters. We then obtained the thermodynamic parameters in the absence of GdnHCl by extrapolating the graph of linear dependence of DeltaH(m) on T(m) to the T(m) corresponding to 0 m GdnHCl. Another key point for checking the validity of our method was the fact that the unfolded state of SBP generated by either heat or GdnHCl is the same by which we could cross-check our results with that obtained from GdnHCl unfolding. Having obtained the true thermodynamic parameters, we report a detailed thermodynamic study of SBP. Further we address the effect of heme in the thermal unfolding mechanism of SBP.     

Contribution of cathepsin L to secretome composition and cleavage pattern of mouse embryonic fibroblasts

The endolysosomal cysteine endoprotease cathepsin L is secreted from cells in a variety of pathological conditions such as cancer and arthritis. We compared the secretome composition and extracellular proteolytic cleavage events in cell supernatants of cathepsin L-deficient and wild-type mouse embryonic fibroblasts (MEFs). Quantitative proteomic comparison of cell conditioned media indicated that cathepsin L deficiency affects, albeit in a limited manner, the abundances of extracellular matrix (ECM) components, signaling proteins, and further proteases as well as endogenous protease inhibitors. Immunodetection corroborated that cathepsin L deficiency results in decreased abundance of the ECM protein periostin and elevated abundance of matrix metalloprotease (MMP)-2. While mRNA levels of MMP-2 were not affected by cathepsin L ablation, periostin mRNA levels were reduced, potentially indicating a downstream effect. To characterize cathepsin L contribution to extracellular proteolysis, we performed terminal amine isotopic labeling of substrates (TAILS), an N-terminomic technique for the identification and quantification of native and proteolytically generated protein N-termini. TAILS identified >1500 protein N-termini. Cathepsin L deficiency predominantly reduced the magnitude of collagenous cleavage sites C-terminal to a proline residue. This contradicts cathepsin L active site specificity and indicates altered activity of further proteases as a result of cathepsin L ablation.     

Antibacterial surfaces based on polymer brushes: investigation on the influence of brush properties on antimicrobial peptide immobilization and antimicrobial activity

Primary amine containing copolymer, poly(N,N-dimethylacrylamide-co-N-(3-aminopropyl)methacrylamide hydrochloride) (poly(DMA-co-APMA)), brushes were synthesized on Ti surface by surface-initiated atom transfer radical polymerization (SI-ATRP) in aqueous conditions. A series of poly(DMA-co-APMA) copolymer brushes on titanium (Ti) surface with different molecular weights, thicknesses, compositions, and graft densities were synthesized by changing the SI-ATRP reaction conditions. Cysteine-functionalized cationic antimicrobial peptide Tet213 (KRWWKWWRRC) was conjugated to the copolymers brushes using a maleimide-thiol addition reaction after initial modification of the grafted chains using 3-maleimidopropionic acid N-hydroxysuccinimide ester. The modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, atomic force microscopy (AFM), and ellipsometry analysis. The conjugation of the Tet213 onto brushes strongly depended on graft density of the brushes at different copolymer brush compositions. The peptide density (peptides/nm(2)) on the surface varied with the initial composition of the copolymer brushes. Higher graft density of the brushes generated high peptide density (pepetide/nm(2)) and lower number of peptides/polymer chain and vice versa. The peptide density and graft density of the chains on surface greatly influenced the antimicrobial activity of peptide grafted polymer brushes against Pseudomonas aeruginosa.     

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia ^1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens ⁴), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs ^{4, 5}. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions³. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers ^{6, 7}, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane ^{8, 9} and mask RBC surface antigens^{10, 11}.     

Late immature mortality is the major influence on reproductive success of African black beetle, Heteronychus arator (Fabricius) (Coleoptera: Scarabaeidae), in a Mediterranean-climate region of Australia

In field and laboratory studies, mortality of African black beetle, Heteronychus arator, in the winter-rainfall, Mediterranean-type climate region of south-western Australia was higher in the late immature stages during summer than in the early immature stages that occur during spring, a contrast to summer-rainfall climatic regions. Greatest mortality occurred around the pupal stage in contrasting soil types, despite drying differences in summer and supplementary watering in some plots. Sampling of natural populations confirmed experimental results that mortality in late immature stages is the major factor limiting H. arator populations under a Mediterranean-type climate. Inter-generation increase in H. arator abundance was uncommon, explaining the consistent abundance typically observed between years in south-western Australia. Random dispersal of newly emerged adults in autumn was inferred to restore uniformity in adult abundance between areas of varying favourability for immature survival.     

Time-resolved Analysis of the Matrix Metalloproteinase 10 Substrate Degradome

Proteolysis is an irreversible post-translational modification that affects intra- and intercellular communication by modulating the activity of bioactive mediators. Key to understanding protease function is the system-wide identification of cleavage events and their dynamics in physiological contexts. Despite recent advances in mass spectrometry-based proteomics for high-throughput substrate screening, current approaches suffer from high false positive rates and only capture single states of protease activity. Here, we present a workflow based on multiplexed terminal amine isotopic labeling of substrates for time-resolved substrate degradomics in complex proteomes. This approach significantly enhances confidence in substrate identification and categorizes cleavage events by specificity and structural accessibility of the cleavage site. We demonstrate concomitant quantification of cleavage site spanning peptides and neo-N and/or neo-C termini to estimate relative ratios of noncleaved and cleaved forms of substrate proteins. By applying this strategy to dissect the matrix metalloproteinase 10 (MMP10) substrate degradome in fibroblast secretomes, we identified the extracellular matrix protein ADAMTS-like protein 1 (ADAMTSL1) as a direct MMP10 substrate and revealed MMP10-dependent ectodomain shedding of platelet-derived growth factor receptor alpha (PDGFRα) as well as sequential processing of type I collagen. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000503.Historically regarded as a mechanism for unspecific degradation of proteins, proteolysis is now recognized as a specific irreversible post-translational modification that affects major intra- and intercellular signaling processes (1, 2). Proteases specifically process bioactive proteins, their receptors, and associated proteins in an interconnected interaction network termed the protease web (3). Dysregulation of the protease web might cause or result from pathologies, such as impaired tissue repair, cancer and neurodegenerative diseases. Therefore, a better understanding of the functions of individual proteases and their interconnections within proteolytic networks is a prerequisite for exploiting proteases as targets for therapeutic intervention (4).To address this issue, several powerful technologies have been developed for the system-wide discovery of protease substrates, i.e. substrate degradomes, in complex and active proteomes (5, 6). A common principle of these mass spectrometry-based methods is the enrichment and monitoring of N-terminal peptides (protein neo-N termini) that are newly generated by a test protease (7). Protein N termini are enriched from complex proteomes either by chemical tagging and affinity resins (positive selection) or by depletion of internal peptides (negative selection) (7). Both principles have been successfully applied in various studies to characterize N-terminomes and to identify protease substrates using in vitro or cell-based systems and more recently also in vivo (8, 9). Negative enrichment approaches were further extended to the analysis of protein C termini (10, 11) and have the general advantage of recording data on naturally blocked (e.g. acetylated) N termini and internal peptides in the same experiment (8).Even if successful in identifying novel proteolytic cleavage events, which could also be validated by orthogonal methods, high-throughput substrate discovery approaches potentially suffer from high numbers of false positive identifications, particularly when employing in vitro systems (12). These have been reduced by monitoring abundances of N-terminal peptides at multiple time points after incubation of a proteome with a test protease (12). In this SILAC-based approach the authors efficiently distinguished critical from bystander cleavages, but it was limited to three time points. Therefore, it did not allow recording kinetic profiles of the relative abundance of N-terminal peptides that are required for determination of apparent kinetic parameters for processing events. Agard et al. elegantly overcame this limitation by use of selected reaction monitoring (SRM)¹ in combination with a positive N-terminal enrichment platform and determined apparent catalytic efficiencies for hundreds of caspase cleavage events in parallel (13). In a similar approach the same group characterized cellular responses to pro-apoptotic cancer drugs by recording time-courses for caspase-generated neo-N termini (14). Although very powerful and highly accurate in quantification, this method strongly exploited the canonical cleavage specificity of caspases after aspartate residues and required a two-stage process involving two types of mass spectrometers. Hence, it would be desirable to monitor the time-resolved generation of neo-N termini in complex proteomes in a single experiment by a simple and robust workflow in an unbiased manner.The development of such an analysis platform would require a reliable method for the system-wide characterization of protein N termini that is easy to perform, fast and highly multiplexible. All these criteria are met by iTRAQ-terminal amine isotopic labeling of substrates (TAILS), a multiplex N-terminome analysis technique that has been applied in 2plex and 4plex experiments to map the matrix metalloproteinase (MMP) 2 and MMP9 substrate degradomes in vitro (15) and most recently to quantitatively analyze the proteome and N-terminome of inflamed mouse skin in the presence or absence of the immune-modulatory protease MMP2 in vivo (8).Here, we exploited the multiplex capabilities of iTRAQ-TAILS by use of 8plex-iTRAQ reagents to monitor the generation of neo-N-terminal peptides by a test protease in complex samples over time. First, using GluC as a test protease with canonical cleavage specificity, we established a workflow for time-resolved substrate degradomics. Recording kinetic profiles significantly increased the confidence in identified cleavage events compared with binary systems and categorized primary cleavage specificities as well as secondary structure elements based on clusters of processing events with different efficiencies. By including data from before N-terminal enrichment, we extended our analysis to neo-C-terminal peptides and concomitantly monitored the generation of neo-N termini and neo-C termini as well as the decrease in abundance of the tryptic peptides spanning the cleavage sites in the same experiment. Next, we applied this approach to the time-resolved analysis of the hardly elucidated substrate degradome of matrix metalloproteinase 10 (MMP10). This important wound- and tumor-related protease is secreted by proliferating and migrating keratinocytes at the wound edge in close proximity to dermal fibroblasts and is also highly expressed in aggressive tumor cells (16–18). Our analysis revealed MMP10-dependent shedding of the platelet-derived growth factor receptor alpha (PDGFRα), processing of ADAMTS-like protein 1 (ADAMTSL1) and multiple cleavages of type I collagen, which could be validated and classified by time-resolved abundance profiles of their corresponding neo-N termini.     

Clinically Approved Iron Chelators Influence Zebrafish Mortality,Hatching Morphology and Cardiac Function

Iron chelation therapy using iron (III) specific chelators such as desferrioxamine (DFO, Desferal), deferasirox (Exjade or ICL-670), and deferiprone (Ferriprox or L1) are the current standard of care for the treatment of iron overload. Although each chelator is capable of promoting some degree of iron excretion, these chelators are also associated with a wide range of well documented toxicities. However, there is currently very limited data available on their effects in developing embryos. In this study, we took advantage of the rapid development and transparency of the zebrafish embryo, Danio rerio to assess and compare the toxicity of iron chelators. All three iron chelators described above were delivered to zebrafish embryos by direct soaking and their effects on mortality, hatching and developmental morphology were monitored for 96 hpf. To determine whether toxicity was specific to embryos, we examined the effects of chelator exposure via intra peritoneal injection on the cardiac function and gene expression in adult zebrafish. Chelators varied significantly in their effects on embryo mortality, hatching and morphology. While none of the embryos or adults exposed to DFO were negatively affected, ICL -treated embryos and adults differed significantly from controls, and L1 exerted toxic effects in embryos alone. ICL-670 significantly increased the mortality of embryos treated with doses of 0.25 mM or higher and also affected embryo morphology, causing curvature of larvae treated with concentrations above 0.5 mM. ICL-670 exposure (10 µL of 0.1 mM injection) also significantly increased the heart rate and cardiac output of adult zebrafish. While L1 exposure did not cause toxicity in adults, it did cause morphological defects in embryos at 0.5 mM. This study provides first evidence on iron chelator toxicity in early development and will help to guide our approach on better understanding the mechanism of iron chelator toxicity.