Phosphoproteomic analysis reveals plant DNA damage signalling pathways with a functional role for histone H2AX phosphorylation in plant growth under genotoxic stress.

DNA damage responses are crucial for plant growth under genotoxic stress. Accumulating evidence indicates that DNA damage responses differ between plant cell types. Here, quantitative shotgun phosphoproteomics provided high-throughput analysis of the DNA damage response network in callus cells. MS analysis revealed a wide network of highly dynamic changes in the phosphoprotein profile of genotoxin-treated cells, largely mediated by the ATAXIA TELANGIECTASIA MUTATED (ATM) protein kinase, representing candidate factors that modulate plant growth, development and DNA repair. A C-terminal dual serine target motif unique to H2AX in the plant lineage showed 171-fold phosphorylation that was absent in atm mutant lines. The physiological significance of post-translational DNA damage signalling to plant growth and survival was demonstrated using reverse genetics and complementation studies of h2ax mutants, establishing the functional role of ATM-mediated histone modification in plant growth under genotoxic stress. Our findings demonstrate the complexity and functional significance of post-translational DNA damage signalling responses in plants and establish the requirement of H2AX phosphorylation for plant survival under genotoxic stress.

A role for repressive complexes and H3K9 di-methylation in PRDM5-associated brittle cornea syndrome.

Type 2 brittle cornea syndrome (BCS2) is an inherited connective tissue disease with a devastating ocular phenotype caused by mutations in the transcription factor PR domain containing 5 (PRDM5) hypothesized to exert epigenetic effects through histone and DNA methylation. Here we investigate clinical samples, including skin fibroblasts and retinal tissue from BCS2 patients, to elucidate the epigenetic role of PRDM5 and mechanisms of its dysregulation in disease. First we report abnormal retinal vascular morphology in the eyes of two cousins with BCS2 (PRDM5 Δ exons 9-14) using immunohistochemistry, and mine data from skin fibroblast expression microarrays from patients with PRDM5 mutations p.Arg590* and Δ exons 9-14, as well as from a PRDM5 ChIP-sequencing experiment. Gene ontology analysis of dysregulated PRDM5-target genes reveals enrichment for extracellular matrix (ECM) genes supporting vascular integrity and development. Q-PCR and ChIP-qPCR confirm upregulation of critical mediators of ECM stability in vascular structures (COL13A1, COL15A1, NTN1, CDH5) in patient fibroblasts. We identify H3K9 di-methylation (H3K9me2) at these PRDM5-target genes in fibroblasts, and demonstrate that the BCS2 mutation p.Arg83Cys diminishes interaction of PRDM5 with repressive complexes, including NuRD complex protein CHD4, and the repressive chromatin interactor HP1BP3, by co-immunoprecipitation combined with mass spectrometry. We observe reduced heterochromatin protein 1 binding protein 3 (HP1BP3) staining in the retinas of two cousins lacking exons 9-14 by immunohistochemistry, and dysregulated H3K9me2 in skin fibroblasts of three patients (p.Arg590*, p.Glu134* and Δ exons 9-14) by western blotting. These findings suggest that defective interaction of PRDM5 with repressive complexes, and dysregulation of H3K9me2, play a role in PRDM5-associated disease.

The mzIdentML data standard for mass spectrometry-based proteomics results.

We report the release of mzIdentML, an exchange standard for peptide and protein identification data, designed by the Proteomics Standards Initiative. The format was developed by the Proteomics Standards Initiative in collaboration with instrument and software vendors, and the developers of the major open-source projects in proteomics. Software implementations have been developed to enable conversion from most popular proprietary and open-source formats, and mzIdentML will soon be supported by the major public repositories. These developments enable proteomics scientists to start working with the standard for exchanging and publishing data sets in support of publications and they provide a stable platform for bioinformatics groups and commercial software vendors to work with a single file format for identification data.

Giant worm-shaped ESCRT scaffolds surround actin-independent integrin clusters.

Endosomal Sorting Complex Required for Transport (ESCRT) proteins can be transiently recruited to the plasma membrane for membrane repair and formation of extracellular vesicles. Here, we discovered micrometer-sized worm-shaped ESCRT structures that stably persist for multiple hours at the plasma membrane of macrophages, dendritic cells, and fibroblasts. These structures surround clusters of integrins and known cargoes of extracellular vesicles. The ESCRT structures are tightly connected to the cellular support and are left behind by the cells together with surrounding patches of membrane. The phospholipid composition is altered at the position of the ESCRT structures, and the actin cytoskeleton is locally degraded, which are hallmarks of membrane damage and extracellular vesicle formation. Disruption of actin polymerization increased the formation of the ESCRT structures and cell adhesion. The ESCRT structures were also present at plasma membrane contact sites with membrane-disrupting silica crystals. We propose that the ESCRT proteins are recruited to adhesion-induced membrane tears to induce extracellular shedding of the damaged membrane.

Proteomic analysis of extracellular matrix from the hepatic stellate cell line LX-2 identifies CYR61 and Wnt-5a as novel constituents of fibrotic liver.

Activation of hepatic stellate cells (HSCs) and subsequent uncontrolled accumulation of altered extracellular matrix (ECM) underpin liver fibrosis, a wound healing response to chronic injury, which can lead to organ failure and death. We sought to catalogue the components of fibrotic liver ECM to obtain insights into disease etiology and aid identification of new biomarkers. Cell-derived ECM was isolated from the HSC line LX-2, an in vitro model of liver fibrosis, and compared to ECM from human foreskin fibroblasts (HFFs) as a control. Mass spectrometry analyses of cell-derived ECMs identified, with ≥99% confidence, 61 structural ECM or secreted proteins (48 and 31 proteins for LX-2 and HFF, respectively). Gene ontology enrichment analysis confirmed the enrichment of ECM proteins, and hierarchical clustering coupled with protein-protein interaction network analysis revealed a subset of proteins enriched to fibrotic ECM, highlighting the existence of cell type-specific ECM niches. Thirty-six proteins were enriched to LX-2 ECM as compared to HFF ECM, of which Wnt-5a and CYR61 were validated by immunohistochemistry in human and murine fibrotic liver tissue. Future studies will determine if these and other components may play a role in the etiology of hepatic fibrosis, serve as novel disease biomarkers, or open up new avenues for drug discovery.

Identifying eIF4E-binding protein translationally-controlled transcripts reveals links to mRNAs bound by specific PUF proteins.

eIF4E-binding proteins (4E-BPs) regulate translation of mRNAs in eukaryotes. However the extent to which specific mRNA targets are regulated by 4E-BPs remains unknown. We performed translational profiling by microarray analysis of polysome and monosome associated mRNAs in wild-type and mutant cells to identify mRNAs in yeast regulated by the 4E-BPs Caf20p and Eap1p; the first-global comparison of 4E-BP target mRNAs. We find that yeast 4E-BPs modulate the translation of >1000 genes. Most target mRNAs differ between the 4E-BPs revealing mRNA specificity for translational control by each 4E-BP. This is supported by observations that eap1Δ and caf20Δ cells have different nitrogen source utilization defects, implying different mRNA targets. To account for the mRNA specificity shown by each 4E-BP, we found correlations between our data sets and previously determined targets of yeast mRNA-binding proteins. We used affinity chromatography experiments to uncover specific RNA-stabilized complexes formed between Caf20p and Puf4p/Puf5p and between Eap1p and Puf1p/Puf2p. Thus the combined action of each 4E-BP with specific 3'-UTR-binding proteins mediates mRNA-specific translational control in yeast, showing that this form of translational control is more widely employed than previously thought.

Zebrafish drug screening identifies candidate therapies for neuroprotection after spontaneous intracerebral haemorrhage.

Despite the global health burden, treatment of spontaneous intracerebral haemorrhage (ICH) is largely supportive, and translation of specific medical therapies has not been successful. Zebrafish larvae offer a unique platform for drug screening to rapidly identify neuroprotective compounds following ICH. We applied the Spectrum Collection library compounds to zebrafish larvae acutely after ICH to screen for decreased brain cell death and identified 150 successful drugs. Candidates were then evaluated for possible indications with other cardiovascular diseases. Six compounds were identified, including two angiotensin-converting enzyme inhibitors (ACE-Is). Ramipril and quinapril were further assessed to confirm a significant 55% reduction in brain cell death. Proteomic analysis revealed potential mechanisms of neuroprotection. Using the INTERACT2 clinical trial dataset, we demonstrated a significant reduction in the adjusted odds of an unfavourable shift in the modified Rankin scale at 90 days for patients receiving an ACE-I after ICH (versus no ACE-I; odds ratio, 0.80; 95% confidence interval, 0.68-0.95; P=0.009). The zebrafish larval model of spontaneous ICH can be used as a reliable drug screening platform and has identified therapeutics that may offer neuroprotection. This article has an associated First Person interview with the first author of the paper.

ISPIDER Central: an integrated database web-server for proteomics.

Despite the growing volumes of proteomic data, integration of the underlying results remains problematic owing to differences in formats, data captured, protein accessions and services available from the individual repositories. To address this, we present the ISPIDER Central Proteomic Database search (http://www.ispider.manchester.ac.uk/cgi-bin/ProteomicSearch.pl), an integration service offering novel search capabilities over leading, mature, proteomic repositories including PRoteomics IDEntifications database (PRIDE), PepSeeker, PeptideAtlas and the Global Proteome Machine. It enables users to search for proteins and peptides that have been characterised in mass spectrometry-based proteomics experiments from different groups, stored in different databases, and view the collated results with specialist viewers/clients. In order to overcome limitations imposed by the great variability in protein accessions used by individual laboratories, the European Bioinformatics Institute's Protein Identifier Cross-Reference (PICR) service is used to resolve accessions from different sequence repositories. Custom-built clients allow users to view peptide/protein identifications in different contexts from multiple experiments and repositories, as well as integration with the Dasty2 client supporting any annotations available from Distributed Annotation System servers. Further information on the protein hits may also be added via external web services able to take a protein as input. This web server offers the first truly integrated access to proteomics repositories and provides a unique service to biologists interested in mass spectrometry-based proteomics.

Upstream sequence elements direct post-transcriptional regulation of gene expression under stress conditions in yeast.

BACKGROUND: The control of gene expression in eukaryotic cells occurs both transcriptionally and post-transcriptionally. Although many genes are now known to be regulated at the translational level, in general, the mechanisms are poorly understood. We have previously presented polysomal gradient and array-based evidence that translational control is widespread in a significant number of genes when yeast cells are exposed to a range of stresses. Here we have re-examined these gene sets, considering the role of UTR sequences in the translational responses of these genes using recent large-scale datasets which define 5' and 3' transcriptional ends for many yeast genes. In particular, we highlight the potential role of 5' UTRs and upstream open reading frames (uORFs). RESULTS: We show a highly significant enrichment in specific GO functional classes for genes that are translationally up- and down-regulated under given stresses (e.g. carbohydrate metabolism is up-regulated under amino acid starvation). Cross-referencing these data with the stress response data we show that translationally upregulated genes have longer 5' UTRs, consistent with their role in translational regulation. In the first genome-wide study of uORFs in a set of mapped 5' UTRs, we show that uORFs are rare, being statistically under-represented in UTR sequences. However, they have distinct compositional biases consistent with their putative role in translational control and are more common in genes which are apparently translationally up-regulated. CONCLUSION: These results demonstrate a central regulatory role for UTR sequences, and 5' UTRs in particular, highlighting the significant role of uORFs in post-transcriptional control in yeast. Yeast uORFs are more highly conserved than has been suggested, lending further weight to their significance as functional elements involved in gene regulation. It also suggests a more complex and novel mechanism of control, whereby uORFs permit genes to escape from a more general attenuation of translation under conditions of stress. However, since uORFs are relatively rare (only ~13% of yeast genes have them) there remain many unanswered questions as to how UTR elements can direct translational control of many hundreds of genes under stress.

Global gene expression profiling reveals widespread yet distinctive translational responses to different eukaryotic translation initiation factor 2B-targeting stress pathways.

Global inhibition of protein synthesis is a hallmark of many cellular stress conditions. Even though specific mRNAs defy this (e.g., yeast GCN4 and mammalian ATF4), the extent and variation of such resistance remain uncertain. In this study, we have identified yeast mRNAs that are translationally maintained following either amino acid depletion or fusel alcohol addition. Both stresses inhibit eukaryotic translation initiation factor 2B, but via different mechanisms. Using microarray analysis of polysome and monosome mRNA pools, we demonstrate that these stress conditions elicit widespread yet distinct translational reprogramming, identifying a fundamental role for translational control in the adaptation to environmental stress. These studies also highlight the complex interplay that exists between different stages in the gene expression pathway to allow specific preordained programs of proteome remodeling. For example, many ribosome biogenesis genes are coregulated at the transcriptional and translational levels following amino acid starvation. The transcriptional regulation of these genes has recently been connected to the regulation of cellular proliferation, and on the basis of our results, the translational control of these mRNAs should be factored into this equation.

PepSeeker: mining information from proteomic data.

Driven by advances in mass spectrometry and analytical chemistry, coupled with the expanding number of completely sequenced genomes, proteomics is becoming a widely exploited technology for characterizing the proteins found in living systems. As proteomics becomes increasingly more high-throughput there is a parallel need for storage of the large quantities of data generated, to support data exchange and allow further analyses. The capture and storage of such data, along with subsequent release and dissemination, not only aid in sharing of the data throughout the proteomics community but also provide scientific insights into the observations between different laboratories, instruments, and software. Growing numbers of resources offer a range of approaches for the capture, storage, and dissemination of proteomic experimental data reflecting the fact that proteomics has now come of age in the postgenomic era and is delivering large, complex datasets that are rich in information. This chapter demonstrates how one such resource, PepSeeker, can be used to mine useful information from proteomic data, which can then be exploited for peptide identification algorithms via a better understanding of how peptides fragment inside mass spectrometers.

PepSeeker: a database of proteome peptide identifications for investigating fragmentation patterns.

Proteome science relies on bioinformatics tools to characterize proteins via their proteolytic peptides which are identified via characteristic mass spectra generated after their ions undergo fragmentation in the gas phase within the mass spectrometer. The resulting secondary ion mass spectra are compared with protein sequence databases in order to identify the amino acid sequence. Although these search tools (e.g. SEQUEST, Mascot, X!Tandem, Phenyx) are frequently successful, much is still not understood about the amino acid sequence patterns which promote/protect particular fragmentation pathways, and hence lead to the presence/absence of particular ions from different ion series. In order to advance this area, we have developed a database, PepSeeker (http://nwsr.smith.man.ac.uk/pepseeker), which captures this peptide identification and ion information from proteome experiments. The database currently contains >185,000 peptides and associated database search information. Users may query this resource to retrieve peptide, protein and spectral information based on protein or peptide information, including the amino acid sequence itself represented by regular expressions coupled with ion series information. We believe this database will be useful to proteome researchers wishing to understand gas phase peptide ion chemistry in order to improve peptide identification strategies. Questions can be addressed to j.selley@manchester.ac.uk.

Dynamic Acclimation to High Light in Arabidopsis thaliana Involves Widespread Reengineering of the Leaf Proteome.

Leaves of Arabidopsis thaliana transferred from low to high light increase their capacity for photosynthesis, a process of dynamic acclimation. A mutant, gpt2, lacking a chloroplast glucose-6-phosphate/phosphate translocator, is deficient in its ability to acclimate to increased light. Here, we have used a label-free proteomics approach, to perform relative quantitation of 1993 proteins from Arabidopsis wild type and gpt2 leaves exposed to increased light. Data are available via ProteomeXchange with identifier PXD006598. Acclimation to light is shown to involve increases in electron transport and carbon metabolism but no change in the abundance of photosynthetic reaction centers. The gpt2 mutant shows a similar increase in total protein content to wild type but differences in the extent of change of certain proteins, including in the relative abundance of the cytochrome b6f complex and plastocyanin, the thylakoid ATPase and selected Benson-Calvin cycle enzymes. Changes in leaf metabolite content as plants acclimate can be explained by changes in the abundance of enzymes involved in metabolism, which were reduced in gpt2 in some cases. Plants of gpt2 invest more in stress-related proteins, suggesting that their reduced ability to acclimate photosynthetic capacity results in increased stress.

Defining the phospho-adhesome through the phosphoproteomic analysis of integrin signalling.

Cell-extracellular matrix (ECM) adhesion is a fundamental requirement for multicellular existence due to roles in positioning, proliferation and differentiation. Phosphorylation plays a major role in adhesion signalling; however, a full understanding of the phosphorylation events that occur at sites of adhesion is lacking. Here we report a proteomic and phosphoproteomic analysis of adhesion complexes isolated from cells spread on fibronectin. We identify 1,174 proteins, 499 of which are phosphorylated (1,109 phosphorylation sites), including both well-characterized and novel adhesion-regulated phosphorylation events. Immunoblotting suggests that two classes of phosphorylated residues are found at adhesion sites-those induced by adhesion and those constitutively phosphorylated but recruited in response to adhesion. Kinase prediction analysis identifies novel kinases with putative roles in adhesion signalling including CDK1, inhibition of which reduces adhesion complex formation. This phospho-adhesome data set constitutes a valuable resource to improve our understanding of the signalling mechanisms through which cell-ECM interactions control cell behaviour.

Analysis of the cartilage proteome from three different mouse models of genetic skeletal diseases reveals common and discrete disease signatures.

Pseudoachondroplasia and multiple epiphyseal dysplasia are genetic skeletal diseases resulting from mutations in cartilage structural proteins. Electron microscopy and immunohistochemistry previously showed that the appearance of the cartilage extracellular matrix (ECM) in targeted mouse models of these diseases is disrupted; however, the precise changes in ECM organization and the pathological consequences remain unknown. Our aim was to determine the effects of matrilin-3 and COMP mutations on the composition and extractability of ECM components to inform how these detrimental changes might influence cartilage organization and degeneration. Cartilage was sequentially extracted using increasing denaturants and the extraction profiles of specific proteins determined using SDS-PAGE/Western blotting. Furthermore, the relative composition of protein pools was determined using mass spectrometry for a non-biased semi-quantitative analysis. Western blotting revealed changes in the extraction of matrilins, COMP and collagen IX in mutant cartilage. Mass spectrometry confirmed quantitative changes in the extraction of structural and non-structural ECM proteins, including proteins with roles in cellular processes such as protein folding and trafficking. In particular, genotype-specific differences in the extraction of collagens XII and XIV and tenascins C and X were identified; interestingly, increased expression of several of these genes has recently been implicated in susceptibility and/or progression of murine osteoarthritis. We demonstrated that mutation of matrilin-3 and COMP caused changes in the extractability of other cartilage proteins and that proteomic analyses of Matn3 V194D, Comp T585M and Comp DelD469 mouse models revealed both common and discrete disease signatures that provide novel insight into skeletal disease mechanisms and cartilage degradation.

Glucose depletion inhibits translation initiation via eIF4A loss and subsequent 48S preinitiation complex accumulation, while the pentose phosphate pathway is coordinately up-regulated.

Cellular stress can globally inhibit translation initiation, and glucose removal from yeast causes one of the most dramatic effects in terms of rapidity and scale. Here we show that the same rapid inhibition occurs during yeast growth as glucose levels diminish. We characterize this novel regulation showing that it involves alterations within the 48S preinitiation complex. In particular, the interaction between eIF4A and eIF4G is destabilized, leading to a temporary stabilization of the eIF3-eIF4G interaction on the 48S complex. Under such conditions, specific mRNAs that are important for the adaptation to the new conditions must continue to be translated. We have determined which mRNAs remain translated early after glucose starvation. These experiments enable us to provide a physiological context for this translational regulation by ascribing defined functions that are translationally maintained or up-regulated. Overrepresented in this class of mRNA are those involved in carbohydrate metabolism, including several mRNAs from the pentose phosphate pathway. Our data support a hypothesis that a concerted preemptive activation of the pentose phosphate pathway, which targets both mRNA transcription and translation, is important for the transition from fermentative to respiratory growth in yeast.

Global translational responses to oxidative stress impact upon multiple levels of protein synthesis.

Global inhibition of protein synthesis is a common response to stress conditions. We have analyzed the regulation of protein synthesis in response to oxidative stress induced by exposure to H(2)O(2) in the yeast Saccharomyces cerevisiae. Our data show that H(2)O(2) causes an inhibition of translation initiation dependent on the Gcn2 protein kinase, which phosphorylates the alpha-subunit of eukaryotic initiation factor-2. Additionally, our data indicate that translation is regulated in a Gcn2-independent manner because protein synthesis was still inhibited in response to H(2)O(2) in a gcn2 mutant. Polysome analysis indicated that H(2)O(2) causes a slower rate of ribosomal runoff, consistent with an inhibitory effect on translation elongation or termination. Furthermore, analysis of ribosomal transit times indicated that oxidative stress increases the average mRNA transit time, confirming a post-initiation inhibition of translation. Using microarray analysis of polysome- and monosome-associated mRNA pools, we demonstrate that certain mRNAs, including mRNAs encoding stress protective molecules, increase in association with ribosomes following H(2)O(2) stress. For some candidate mRNAs, we show that a low concentration of H(2)O(2) results in increased protein production. In contrast, a high concentration of H(2)O(2) promotes polyribosome association but does not necessarily lead to increased protein production. We suggest that these mRNAs may represent an mRNA store that could become rapidly activated following relief of the stress condition. In summary, oxidative stress elicits complex translational reprogramming that is fundamental for adaptation to the stress.