A phospho-dawn of protein modification anticipates light onset in the picoeukaryote Ostreococcus tauri.

Diel regulation of protein levels and protein modification had been less studied than transcript rhythms. Here, we compare transcriptome data under light-dark cycles with partial proteome and phosphoproteome data, assayed using shotgun MS, from the alga Ostreococcus tauri, the smallest free-living eukaryote. A total of 10% of quantified proteins but two-thirds of phosphoproteins were rhythmic. Mathematical modelling showed that light-stimulated protein synthesis can account for the observed clustering of protein peaks in the daytime. Prompted by night-peaking and apparently dark-stable proteins, we also tested cultures under prolonged darkness, where the proteome changed less than under the diel cycle. Among the dark-stable proteins were prasinophyte-specific sequences that were also reported to accumulate when O. tauri formed lipid droplets. In the phosphoproteome, 39% of rhythmic phospho-sites reached peak levels just before dawn. This anticipatory phosphorylation suggests that a clock-regulated phospho-dawn prepares green cells for daytime functions. Acid-directed and proline-directed protein phosphorylation sites were regulated in antiphase, implicating the clock-related casein kinases 1 and 2 in phase-specific regulation, alternating with the CMGC protein kinase family. Understanding the dynamic phosphoprotein network should be facilitated by the minimal kinome and proteome of O. tauri. The data are available from ProteomeXchange, with identifiers PXD001734, PXD001735, and PXD002909.

Surviving Starvation: Proteomic and Lipidomic Profiling of Nutrient Deprivation in the Smallest Known Free-Living Eukaryote.

Marine phytoplankton, comprising cyanobacteria, micro- and pico-algae are key to photosynthesis, oxygen production and carbon assimilation on Earth. The unicellular green picoalga Ostreococcus tauri holds a key position at the base of the green lineage of plants, which makes it an interesting model organism. O. tauri has adapted to survive in low levels of nitrogen and phosphorus in the open ocean and also during rapid changes in the levels of these nutrients in coastal waters. In this study, we have employed untargeted proteomic and lipidomic strategies to investigate the molecular responses of O. tauri to low-nitrogen and low-phosphorus environments. In the absence of external nitrogen, there was an elevation in the expression of ammonia and urea transporter proteins together with an accumulation of triglycerides. In phosphate-limiting conditions, the expression levels of phosphokinases and phosphate transporters were increased, indicating an attempt to maximise scavenging opportunities as opposed to energy conservation conditions. The production of betaine lipids was also elevated, highlighting a shift away from phospholipid metabolism. This finding was supported by the putative identification of betaine synthase in O. tauri. This work offers additional perspectives on the complex strategies that underpin the adaptive processes of the smallest known free-living eukaryote to alterations in environmental conditions.

Proteomic profiling of cellular steatosis with concomitant oxidative stress in vitro.

BACKGROUND: Nutrient excess underpins the development of nonalcoholic fatty liver disease (NAFLD). The ensuing metabolic derangement is characterised by increased cellular respiration, oxidative stress and mitochondrial impairment. We have previously recapitulated these events in an in vitro cellular steatosis model. Here, we examined the distinct patterns of protein expression involved using a proteomics approach. METHODS: Human hepatoblastoma C3A cells were treated with a combination of energy substrates; lactate (L), pyruvate (P), octanoate (O) and ammonia (N). Proteins extracts were trypsinized and analyzed on a capillary HPLC OrbitrapXL mass spectrometer. Proteins were quantified using a label-free intensity based approach. Functional enrichment analysis was performed using ToppCluster via Gene Ontology (GO) database. RESULTS: Of the 1327 proteins identified, 104 were differentially expressed between LPON and untreated cells (defined as: ≥2 peptides; fold change ≥1.5; p-value <0.05). Seventy of these were upregulated with LPON. Functional enrichment analysis revealed enhanced protein biosynthesis accompanied by downregulation of histones H2A type 1-A, H1.2, H1.5 and H1.0I in LPON cells. Lipid binding annotations were also enriched as well as proteins involved in cholesterol synthesis, uptake and efflux. Increased expression of aldo-keto reductase family 1, member C1 and C3 suggests enhanced sterol metabolism and increased ROS-mediated lipid peroxidation. CONCLUSIONS: The surge of energy substrates diverts free fatty acid metabolism towards pathways that can mitigate lipotoxicity. The histones depletion may represent an adaptation to increased protein synthesis. However, this can also expose DNA to oxidative stress thus should be explored further in the context of NAFLD progression.

Rock geochemistry induces stress and starvation responses in the bacterial proteome.

Interactions between microorganisms and rocks play an important role in Earth system processes. However, little is known about the molecular capabilities microorganisms require to live in rocky environments. Using a quantitative label-free proteomics approach, we show that a model bacterium (Cupriavidus metallidurans CH34) can use volcanic rock to satisfy some elemental requirements, resulting in increased rates of cell division in both magnesium- and iron-limited media. However, the rocks also introduced multiple new stresses via chemical changes associated with pH, elemental leaching and surface adsorption of nutrients that were reflected in the proteome. For example, the loss of bioavailable phosphorus was observed and resulted in the upregulation of diverse phosphate limitation proteins, which facilitate increase phosphate uptake and scavenging within the cell. Our results revealed that despite the provision of essential elements, rock chemistry drives complex metabolic reorganization within rock-dwelling organisms, requiring tight regulation of cellular processes at the protein level. This study advances our ability to identify key microbial responses that enable life to persist in rock environments.

Fibrinogen production is enhanced in an in-vitro model of non-alcoholic fatty liver disease: an isolated risk factor for cardiovascular events?

BACKGROUND: Cardiovascular disease (CVD) remains the major cause of excess mortality in patients with non-alcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the individual contribution of NAFLD to CVD risk factors in the absence of pathogenic influences from other comorbidities often found in NAFLD patients, by using an established in-vitro model of hepatic steatosis. METHODS: Histopathological events in non-alcoholic fatty liver disease were recapitulated by focused metabolic nutrient overload of hepatoblastoma C3A cells, using oleate-treated-cells and untreated controls for comparison. Microarray and proteomic data from cell culture experiments were integrated into a custom-built systems biology database and proteogenomics analysis performed. Candidate genes with significant dysregulation and concomitant changes in protein abundance were identified and STRING association and enrichment analysis performed to identify putative pathogenic pathways. RESULTS: The search strategy yielded 3 candidate genes that were specifically and significantly up-regulated in nutrient-overloaded cells compared to untreated controls: fibrinogen alpha chain (2.2 fold), fibrinogen beta chain (2.3 fold) and fibrinogen gamma chain (2.1 fold) (all rank products pfp <0.05). Fibrinogen alpha and gamma chain also demonstrated significant concomitant increases in protein abundance (3.8-fold and 2.0-fold, respectively, p <0.05). CONCLUSIONS: In-vitro modelling of NAFLD and reactive oxygen species formation in nutrient overloaded C3A cells, in the absence of pathogenic influences from other comorbidities, suggests that NAFLD is an isolated determinant of CVD. Nutrient overload-induced up-regulation of all three fibrinogen component subunits of the coagulation cascade provides a possible mechanism to explain the excess CVD mortality observed in NAFLD patients.

Label-free quantitative analysis of the casein kinase 2-responsive phosphoproteome of the marine minimal model species Ostreococcus tauri.

Casein kinase 2 (CK2) is a protein kinase that phosphorylates a plethora of cellular target proteins involved in processes including DNA repair, cell cycle control, and circadian timekeeping. CK2 is functionally conserved across eukaryotes, although the substrate proteins identified in a range of complex tissues are often different. The marine alga Ostreococcus tauri is a unicellular eukaryotic model organism ideally suited to efficiently study generic roles of CK2 in the cellular circadian clock. Overexpression of CK2 leads to a slow circadian rhythm, verifying functional conservation of CK2 in timekeeping. The proteome was analysed in wild-type and CK2-overexpressing algae at dawn and dusk, revealing that differential abundance of the global proteome across the day is largely unaffected by overexpression. However, CK2 activity contributed more strongly to timekeeping at dusk than at dawn. The phosphoproteome of a CK2 overexpression line and cells treated with CK2 inhibitor was therefore analysed and compared to control cells at dusk. We report an extensive catalogue of 447 unique CK2-responsive differential phosphopeptide motifs to inform future studies into CK2 activity in the circadian clock of more complex tissues. All MS data have been deposited in the ProteomeXchange with identifier PXD000975 (http://proteomecentral.proteomexchange.org/dataset/PXD000975).

Sample preparation for phosphoproteomic analysis of circadian time series in Arabidopsis thaliana.

Systems biological approaches to study the Arabidopsis thaliana circadian clock have mainly focused on transcriptomics while little is known about the proteome, and even less about posttranslational modifications. Evidence has emerged that posttranslational protein modifications, in particular phosphorylation, play an important role for the clock and its output. Phosphoproteomics is the method of choice for a large-scale approach to gain more knowledge about rhythmic protein phosphorylation. Recent plant phosphoproteomics publications have identified several thousand phosphopeptides. However, the methods used in these studies are very labor-intensive and therefore not suitable to apply to a well-replicated circadian time series. To address this issue, we present and compare different strategies for sample preparation for phosphoproteomics that are compatible with large numbers of samples. Methods are compared regarding number of identifications, variability of quantitation, and functional categorization. We focus on the type of detergent used for protein extraction as well as methods for its removal. We also test a simple two-fraction separation of the protein extract.

The reduced kinome of Ostreococcus tauri: core eukaryotic signalling components in a tractable model species.

BACKGROUND: The current knowledge of eukaryote signalling originates from phenotypically diverse organisms. There is a pressing need to identify conserved signalling components among eukaryotes, which will lead to the transfer of knowledge across kingdoms. Two useful properties of a eukaryote model for signalling are (1) reduced signalling complexity, and (2) conservation of signalling components. The alga Ostreococcus tauri is described as the smallest free-living eukaryote. With less than 8,000 genes, it represents a highly constrained genomic palette. RESULTS: Our survey revealed 133 protein kinases and 34 protein phosphatases (1.7% and 0.4% of the proteome). We conducted phosphoproteomic experiments and constructed domain structures and phylogenies for the catalytic protein-kinases. For each of the major kinases families we review the completeness and divergence of O. tauri representatives in comparison to the well-studied kinomes of the laboratory models Arabidopsis thaliana and Saccharomyces cerevisiae, and of Homo sapiens. Many kinase clades in O. tauri were reduced to a single member, in preference to the loss of family diversity, whereas TKL and ABC1 clades were expanded. We also identified kinases that have been lost in A. thaliana but retained in O. tauri. For three, contrasting eukaryotic pathways - TOR, MAPK, and the circadian clock - we established the subset of conserved components and demonstrate conserved sites of substrate phosphorylation and kinase motifs. CONCLUSIONS: We conclude that O. tauri satisfies our two central requirements. Several of its kinases are more closely related to H. sapiens orthologs than S. cerevisiae is to H. sapiens. The greatly reduced kinome of O. tauri is therefore a suitable model for signalling in free-living eukaryotes.

The proteomic response in glioblastoma in young patients.

Increasing age is an important prognostic variable in glioblastoma (GBM). We have defined the proteomic response in GBM samples from 7 young patients (mean age 36 years) compared to peritumoural-control samples from 10 young patients (mean age 32 years). 2-Dimensional-gel-electrophoresis, image analysis, and protein identification (LC/MS) were performed. 68 proteins were significantly altered in young GBM samples with 29 proteins upregulated and 39 proteins downregulated. Over 50 proteins are described as altered in GBM for the first time. In a parallel analysis in old GBM (mean age 67 years), an excellent correlation could be demonstrated between the proteomic profile in young GBM and that in old GBM patients (r(2) = 0.95) with only 5 proteins altered significantly (p < 0.01). The proteomic response in young GBM patients highlighted alterations in protein-protein interactions in the immunoproteosome, NFkB signalling, and mitochondrial function and the same systems participated in the responses in old GBM patients.

Interactions among mitochondrial proteins altered in glioblastoma.

Mitochondrial dysfunction is putatively central to glioblastoma (GBM) pathophysiology but there has been no systematic analysis in GBM of the proteins which are integral to mitochondrial function. Alterations in proteins in mitochondrial enriched fractions from patients with GBM were defined with label-free liquid chromatography mass spectrometry. 256 mitochondrially-associated proteins were identified in mitochondrial enriched fractions and 117 of these mitochondrial proteins were markedly (fold-change ≥ 2) and significantly altered in GBM (p ≤ 0.05). Proteins associated with oxidative damage (including catalase, superoxide dismutase 2, peroxiredoxin 1 and peroxiredoxin 4) were increased in GBM. Protein-protein interaction analysis highlighted a reduction in multiple proteins coupled to energy metabolism (in particular respiratory chain proteins, including 23 complex-I proteins). Qualitative ultrastructural analysis in GBM with electron microscopy showed a notably higher prevalence of mitochondria with cristolysis in GBM. This study highlights the complex mitochondrial proteomic adjustments which occur in GBM pathophysiology.

Functional analysis of the rodent CK1tau mutation in the circadian clock of a marine unicellular alga.

BACKGROUND: Casein Kinase 1 (CK1) is one of few proteins known to affect cellular timekeeping across metazoans, and the naturally occurring CK1tau mutation shortens circadian period in mammals. Functional conservation of a timekeeping function for CK1 in the green lineage was recently identified in the green marine unicell Ostreococcus tauri, in spite of the absence of CK1's transcriptional targets known from other species. The short-period phenotype of CK1tau mutant in mammals depends specifically on increased CK1 activity against PERIOD proteins. To understand how CK1 acts differently upon the algal clock, we analysed the cellular and proteomic effects of CK1tau overexpression in O. tauri. RESULTS: Overexpression of the CK1tau in O. tauri induces period lengthening identical to overexpression of wild-type CK1, in addition to resistance to CK1 inhibitor IC261. Label-free quantitative mass spectrometry of CK1tau overexpressing algae revealed a total of 58 unique phospho-sites that are differentially responsive to CK1tau. Combined with CK1 phosphorylation site prediction tools and previously published wild-type CK1-responsive peptides, this study results in a highly stringent list of upregulated phospho-sites, derived from proteins containing ankyrin repeats, kinase proteins, and phosphoinositide-binding proteins. CONCLUSIONS: The identical phenotype for overexpression of wild-type CK1 and CK1tau is in line with the absence of critical targets for rodent CK1tau in O. tauri. Proteomic analyses reveal that two thirds of previously reported CK1 overexpression-responsive phospho-sites are shared with CK1tau. These results indicate that the two alleles are functionally indiscriminate in O. tauri, and verify the identified cellular CK1 target proteins in a minimal circadian model organism.

Functional analysis of Casein Kinase 1 in a minimal circadian system.

The Earth's rotation has driven the evolution of cellular circadian clocks to facilitate anticipation of the solar cycle. Some evidence for timekeeping mechanism conserved from early unicellular life through to modern organisms was recently identified, but the components of this oscillator are currently unknown. Although very few clock components appear to be shared across higher species, Casein Kinase 1 (CK1) is known to affect timekeeping across metazoans and fungi, but has not previously been implicated in the circadian clock in the plant kingdom. We now show that modulation of CK1 function lengthens circadian rhythms in Ostreococcustauri, a unicellular marine algal species at the base of the green lineage, separated from humans by ~1.5 billion years of evolution. CK1 contributes to timekeeping in a phase-dependent manner, indicating clock-mediated gating of CK1 activity. Label-free proteomic analyses upon overexpression as well as inhibition revealed CK1-responsive phosphorylation events on a set of target proteins, including highly conserved potentially clock-relevant cellular regulator proteins. These results have major implications for our understanding of cellular timekeeping and can inform future studies in any circadian organism.

PROTEINCHALLENGE: crowd sourcing in proteomics analysis and software development.

In large-scale proteomics studies there is a temptation, after months of experimental work, to plug resulting data into a convenient-if poorly implemented-set of tools, which may neither do the data justice nor help answer the scientific question. In this paper we have captured key concerns, including arguments for community-wide open source software development and "big data" compatible solutions for the future. For the meantime, we have laid out ten top tips for data processing. With these at hand, a first large-scale proteomics analysis hopefully becomes less daunting to navigate. However there is clearly a real need for robust tools, standard operating procedures and general acceptance of best practises. Thus we submit to the proteomics community a call for a community-wide open set of proteomics analysis challenges--PROTEINCHALLENGE--that directly target and compare data analysis workflows, with the aim of setting a community-driven gold standard for data handling, reporting and sharing.

Proteome turnover in the green alga Ostreococcus tauri by time course 15N metabolic labeling mass spectrometry.

Protein synthesis and degradation determine the cellular levels of proteins, and their control hence enables organisms to respond to environmental change. Experimentally, these are little known proteome parameters; however, recently, SILAC-based mass spectrometry studies have begun to quantify turnover in the proteomes of cell lines, yeast, and animals. Here, we present a proteome-scale method to quantify turnover and calculate synthesis and degradation rate constants of individual proteins in autotrophic organisms such as algae and plants. The workflow is based on the automated analysis of partial stable isotope incorporation with (15)N. We applied it in a study of the unicellular pico-alga Ostreococcus tauri and observed high relative turnover in chloroplast-encoded ATPases (0.42-0.58% h(-1)), core photosystem II proteins (0.34-0.51% h(-1)), and RbcL (0.47% h(-1)), while nuclear-encoded RbcS2 is more stable (0.23% h(-1)). Mitochondrial targeted ATPases (0.14-0.16% h(-1)), photosystem antennae (0.09-0.14% h(-1)), and histones (0.07-0.1% h(-1)) were comparatively stable. The calculation of degradation and synthesis rate constants k(deg) and k(syn) confirms RbcL as the bulk contributor to overall protein turnover. This study performed over 144 h of incorporation reveals dynamics of protein complex subunits as well as isoforms targeted to different organelles.

Proteomic analysis of mitochondria in APOE transgenic mice and in response to an ischemic challenge.

Apolipoprotein E (APOE)-ɛ4 is associated with a deleterious outcome after ischemic brain injury, which may involve abnormal regulation of mitochondrial function. We have assessed the mitochondrial proteomic response of APOE-ɛ3 and APOE-ɛ4 transgenic mice to transient global ischemic injury in the hippocampus. A genotype-dependent increase in ApoE levels in mitochondria was observed after ischemia, with APOE-ɛ4 mice showing significantly greater increases than APOE-ɛ3 mice. Quantitative analysis of the mitochondria-enriched fractions was performed using liquid-chromatography mass spectrometry coupled to label-free analysis. Of the 1,067 identified proteins, 274 were mitochondria associated. Mitochondrial protein expression was significantly different between genotypes under basal conditions as well as in response to global ischemia. A total of 12 mitochondrial proteins (including respiratory chain proteins NDUFA11, NDUFS3, NDUF5B, ATP5J, as well as ETFA, CYB5B, ATP6V1A, HSPA1B, OXR1, GLUL, IARS2, and PHYHIPL) were significantly altered with respect to genotype, global ischemia, or their interaction (P<0.01). A compelling interactome, created using proteins found to be significantly modulated by global ischemia (P<0.05), involved proteins that regulate energy production and oxidative stress. Thus, APOE genotype has a differential effect on the mitochondrial protein expression in the absence and presence of an injury, which may underlie the differing genotype susceptibility.

Shotgun proteomic analysis of the unicellular alga Ostreococcus tauri.

Ostreococcus tauri is a unicellular green alga and amongst the smallest and simplest free-living eukaryotes. The O. tauri genome sequence was determined in 2006. Molecular, physiological and taxonomic data that has been generated since then highlight its potential as a simple model species for algae and plants. However, its proteome remains largely unexplored. This paper describes the global proteomic study of O. tauri, using mass spectrometry-based approaches: phosphopeptide enrichment, cellular fractionation, label-free quantification and (15)N metabolic labeling. The O. tauri proteome was analyzed under the following conditions: sampling at different times during the circadian cycle, after 24h of illumination, after 24h of darkness and under various nitrogen source supply levels. Cell cycle related proteins such as dynamin and kinesin were significantly up-regulated during the daylight-to-darkness transition. This is reflected by their higher intensity at ZT13 and this transition phase coincides with the end of mitosis. Proteins involved in several metabolic mechanisms were found to be up-regulated under low nitrogen conditions, including carbon storage pathways, glycolysis, phosphate transport, and the synthesis of inorganic polyphosphates. Ostreococcus tauri responds to low nitrogen conditions by reducing its nitrogen assimilation machinery which suggests an atypical adaptation mechanism for coping with a nutrient-limited environment.

Quantitative analysis of multi-protein interactions using FRET: application to the SUMO pathway.

Protein-protein binding and signaling pathways are important fields of biomedical science. Here we report simple optical methods for the determination of the equilibrium binding constant K(d) of protein-protein interactions as well as quantitative studies of biochemical cascades. The techniques are based on steady-state and time-resolved fluorescence resonance energy transfer (FRET) between ECFP and Venus-YFP fused to proteins of the SUMO family. Using FRET has several advantages over conventional free-solution techniques such as isothermal titration calorimetry (ITC): Concentrations are determined accurately by absorbance, highly sensitive binding signals enable the analysis of small quantities, and assays are compatible with multi-well plate format. Most importantly, our FRET-based techniques enable us to measure the effect of other molecules on the binding of two proteins of interest, which is not straightforward with other approaches. These assays provide powerful tools for the study of competitive biochemical cascades and the extent to which drug candidates modify protein interactions.

A fluorescence-resonance-energy-transfer-based protease activity assay and its use to monitor paralog-specific small ubiquitin-like modifier processing.

Dynamic modification of proteins with the small ubiquitin-like modifier (SUMO) affects the stability, cellular localization, enzymatic activity, and molecular interactions of a wide spectrum of protein targets. We have developed an in vitro fluorescence-resonance-energy-transfer-based assay that uses bacterially expressed substrates for the rapid and quantitative analysis of SUMO paralog-specific C-terminal hydrolase activity. This assay has applications in SUMO protease characterization, enzyme kinetic analysis, determination of SUMO protease activity in eukaryotic cell extracts, and high-throughput inhibitor screening. In addition, while demonstrating such uses, we show that the SUMO-1 processing activity in crude HeLa cell extracts is far greater than that of SUMO-2, implying that differential maturation rates of SUMO paralogs in vivo may be functionally significant. The high degree of structural conservation across the ubiquitin-like protein superfamily suggests that the general principle of this assay should be applicable to other post-translational protein modification systems.

Fluorescence spectroscopy of an in vitro model of human cervical precancer identifies neoplastic phenotype.

The clinical diagnosis of cervical neoplasia by spectroscopic methods is potentially a reliable, fast and cost-effective alternative to the conventional smear test. However, it is currently limited by significant inter-patient variation in the spectroscopic properties of the cervix. Characterisation of suitable in vitro models of the spectroscopic changes that take place during neoplastic progression may prove to be a significant step towards the successful development of reliable in vivo systems. In this study, we used organotypic epithelial raft culture as an in vitro model of cervical tissue to analyse changes in the fluorescence properties of surface squamous epithelium that are associated with the development of neoplastic disease. Collagen plugs lined by primary human keratinocytes (PHKs) were used to model the normal cervical epithelium, and plugs lined by cells of the SiHa line were used as a model of neoplastic cervical tissue. Fluorescence emission spectra of these rafts were recorded at excitation wavelengths in the 250-330 nm range, complementing previous work published at longer wavelengths. Normalised, truncated emission spectra were analysed using multivariate principal component analysis. We successfully distinguished between in vitro models of normal and neoplastic cervical tissue and demonstrated a differential effect of acetic acid, which enhances the discrimination of normal from neoplastic tissue. Identification of these differences between in vitro organotypic epithelial rafts may ultimately aid the discrimination of cervical lesions in vivo.