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.

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.

Temporal profiling of the coding and noncoding murine cytomegalovirus transcriptomes.

The global transcriptional program of murine cytomegalovirus (MCMV), involving coding, noncoding, and antisense transcription, remains unknown. Here we report an oligonucleotide custom microarray platform capable of measuring both coding and noncoding transcription on a genome-wide scale. By profiling MCMV wild-type and immediate-early mutant strains in fibroblasts, we found rapid activation of the transcriptome by 6.5 h postinfection, with absolute dependency on ie3, but not ie1 or ie2, for genomic programming of viral gene expression. Evidence is also presented to show, for the first time, genome-wide noncoding and bidirectional transcription at late stages of MCMV infection.

Network generation enhances interpretation of proteomic data from induced apoptosis.

Thirteen proteins (identified with 2-D gels and MALDI-TOF MS) are significantly altered during staurosporine-induced apoptosis in SH-SY5Y cells. To gain further insight into the integrated cellular response to apoptosis, we have investigated whether a network can be generated of direct and indirect interactions between these 13 proteins. A network that contains 12 out of the 13 proteins was generated using Ingenuity Pathway Analysis (IPA) and this network is dominated (89%) by direct protein-protein interactions. This network scored 34 with IPA. Bootstrapping 1000 random lists of 13 proteins suggested that the frequency of this score occurring by chance was 1 in 500. We examined whether subsets of proteins such as HSPs, which were elevated after staurosporine, had a disproportionate impact on the network generated. There was no evidence that any subset of 8 from the 13 proteins contributed disproportionately to the network. Network generation, using IPA, identified common features (such as endoplasmic reticular stress protein interactions) in apoptotic studies from different laboratories. The generation of protein interaction networks clearly enhances the interpretation of proteomic data, but only when interpreted cautiously, particularly in respect of statistical analyses.

The odour span task: a novel paradigm for assessing working memory in mice.

Impoverished odour recognition and memory are amongst the earliest symptoms observed in mild cognitive impairment, Alzheimer's disease and schizophrenia, and have been advocated as early disease bio-markers. Although transgenic animals modelling disease pathologies continually emerge, there remains a paucity of tasks to examine olfactory working memory in mice. The present studies describe a mouse odour span task, which assesses the ability to remember increasing numbers of odours. Since caspase-3 is highly expressed throughout the olfactory system, we postulated that mice over-expressing this apoptogenic protein would exhibit impaired performance in the odour span task. Mice over-expressing human caspase-3 (Tg) exhibited age-independent deficits in olfactory working memory (6-18 months) compared with wild-type littermates, requiring longer for task acquisition and exhibiting impaired asymptotic performance, with reduced span lengths, lower accuracy and increased error rates. These impairments appeared to be selective for working memory, as Tg mice had no deficits in odour discriminatory ability or in locomotor measures. Importantly, nicotine, which improves working memory span in man, reversed the deficits exhibited by Tg mice. In conclusion, the mouse odour span task can detect subtle changes in olfactory working memory induced by genetic manipulation and drug administration and therefore should be applied to animal models of neurological disease.

Impaired attention is central to the cognitive deficits observed in alpha 7 deficient mice.

alpha7-Nicotinic acetylcholine receptors (alpha7-nAChR) have been implicated in a range of cognitive deficits in schizophrenia. Therefore we examined alpha7-nAChR knockout (KO), heterozygote (HT) and wildtype (WT) littermate mice in the 5-CSR (a rodent model of sustained attention) and odour span (a novel mouse working memory paradigm) tasks, and related performance to nAChR density. Whilst there was no difference between groups in baseline 5-CSR task performance, alpha7-nAChR KO's exhibited significantly higher omission levels compared to WT mice on increasing the attentional load, with HT mice performing at an intermediate level. Furthermore, alpha7-nAChR KO mice were significantly impaired in the odour span task when compared to WT mice, in a pattern consistent with impaired attention. These behavioural deficits were associated with the loss of alpha7-nAChRs, as alpha4beta2-nAChR density was unaltered in these mice. Thus these studies intimate that the attentional impairment in alpha7-nAChR transgenic mice maybe core to other deficits in cognition.

Differential profile of Nix upregulation and translocation during hypoxia/ischaemia in vivo versus in vitro.

Nix, a hypoxia-sensitive member of the Bcl-2 family, is upregulated at the mRNA level during hypoxia through induction of a hypoxia-inducible factor-1 alpha (HIF-1 alpha) response element in its promoter sequence. However, the mechanism(s) regulating Nix protein activation remain unclear. The present studies examine Nix protein expression and subcellular distribution in response to hypoxic stimuli in vivo and in culture and to two disparate apoptotic stimuli in vitro. Upregulation and translocation of Nix (by day 5) in hypoxic/serum-deprived CHO-K1 cells, was preceded by Bax activation (by day 4) and caspase-3 processing (by day 2), suggesting that initiation of cell death in vitro is a Nix-independent event. In contrast, an early Nix response (upregulation and translocation to the mitochondria) was observed after 6 h of middle cerebral artery occlusion in the rat. Nix translocation was observed in the ipsilateral cortex and striatum before other histological (infarct development, neuronal loss, apoptotic body formation) or biochemical (Bax activation or caspase-3 cleavage) markers of damage were detected. While fundamental differences between hypoxia/ischaemia in culture and in vivo likely explain the different temporal profiles of Nix, Bax, and caspase-3 activation observed, these studies show that like Bax, mitochondrial accumulation is a common event during Nix activation. These are the first studies to show upregulation and translocation of Nix in the ischaemic brain and suggest Nix to be a novel therapeutic target in ischaemic research. Moreover, Nix upregulation in staurosporine-treated SH-SY5Y cells and dexamethasone-treated A1.1 cells supports a more generalized role for Nix in apoptotic cell death.

Adaptive changes in the neuronal proteome: mitochondrial energy production, endoplasmic reticulum stress, and ribosomal dysfunction in the cellular response to metabolic stress.

Impaired energy metabolism in neurons is integral to a range of neurodegenerative diseases, from Alzheimer's disease to stroke. To investigate the complex molecular changes underpinning cellular adaptation to metabolic stress, we have defined the proteomic response of the SH-SY5Y human neuroblastoma cell line after exposure to a metabolic challenge of oxygen glucose deprivation (OGD) in vitro. A total of 958 proteins across multiple subcellular compartments were detected and quantified by label-free liquid chromatography mass spectrometry. The levels of 130 proteins were significantly increased (P<0.01) after OGD and the levels of 63 proteins were significantly decreased (P<0.01) while expression of the majority of proteins (765) was not altered. Network analysis identified novel protein-protein interactomes involved with mitochondrial energy production, protein folding, and protein degradation, indicative of coherent and integrated proteomic responses to the metabolic challenge. Approximately one third (61) of the differentially expressed proteins was associated with the endoplasmic reticulum and mitochondria. Electron microscopic analysis of these subcellular structures showed morphologic changes consistent with the identified proteomic alterations. Our investigation of the global cellular response to a metabolic challenge clearly shows the considerable adaptive capacity of the proteome to a slowly evolving metabolic challenge.

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.

Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format.

The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated protocols for the extraction of total RNA from 9-day-old Arabidopsis seedlings in a 96 well plate format using silica membrane-based methodology. Consistent and reproducible yields of high quality RNA are isolated averaging 8.9 µg total RNA per sample (~20 mg plant tissue). The purified RNA is suitable for subsequent qPCR analysis of the expression of over 500 genes in triplicate from each sample. Using the automated procedure, 192 samples (2 × 96 well plates) can easily be fully processed (samples homogenised, RNA purified and quantified) in less than half a day. Additionally we demonstrate that plant samples can be stored in RNAlater at -20°C (but not 4°C) for 10 months prior to extraction with no significant effect on RNA yield or quality. Additionally, disrupted samples can be stored in the lysis buffer at -20°C for at least 6 months prior to completion of the extraction procedure providing a flexible sampling and storage scheme to facilitate complex time series experiments.

The use of a novel quantitation strategy based on Reductive Isotopic Di-Ethylation (RIDE) to evaluate the effect of glufosinate on the unicellular algae Ostreococcus tauri.

We report a novel stable-isotope labeling strategy for quantitative proteomics analysis. The method consists of labeling N-termini and lysine ε-amino groups through reductive amination using acetaldehyde. This allows isotope labeling using pairs of either 2H/1H or 13C/12C without mass spectrum overlap. Our labeling procedure, which is significantly different than that developed for dimethylation, can be completed with little trace of partial ethylation; non-labeled peptides represent less than 0.05% of all peptides. Co-elution of both isotopic 13C/12C peptide pairs was observed in all cases, simplifying data analysis, which can be performed using standard commercial software such as Mascot Distiller. A 13C/12C labeled mix in a 1:1 ratio from a complex extract digest of the unicellular algae Ostreococcus tauri, showed a relative standard deviation of less than 14%. This quantitative method was used to characterize O. tauri in the presence of glufosinate, an herbicide which inhibits glutamine synthetase. Blocking glutamine synthetase significantly reduced the expression of several enzymes and transporters involved in nitrogen assimilation and the expression of a number of proteins involved in various stresses including oxidative damage response were up-regulated.

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.

Apoptosis induced by staurosporine alters chaperone and endoplasmic reticulum proteins: Identification by quantitative proteomics.

Apoptosis contributes to cell death after cerebral ischaemia. A quantitative proteomics approach has been employed to define alterations in protein levels in apoptosis induced with staurosporine (STS). Human neuroblastoma derived SH-SY5Y cells were treated with STS (500 nM for 6 h) to induce apoptosis. Quantitative 2-DE was used to determine the changing protein levels with MALDI-TOF MS identification of proteins. Of the 154 proteins analysed, 13 proteins were significantly altered as a result of the apoptotic stimulus; ten of the proteins showed an increase in level with STS and were identified as heat shock cognate 71 (Hsc71), two isoforms of heat shock protein 70 (Hsp70), glucose regulated protein 78 (GRP78), F-actin capping protein, stress-induced phosphoprotein 1, chromatin assembly factor 1 (CAF-1), protein disulphide isomerase A3 (PDI A3) precursor, transitional ER ATPase and actin interacting protein 1 (AIP 1). Three proteins which displayed significant decrease in levels with STS were identified as tubulin, vimentin and glucose regulated protein 94 (GRP94). The functional roles and subcellular locations of these proteins collectively indicate that STS-induced apoptosis provokes induces an unfolded protein response involving molecular chaperones, cochaperones and structural proteins indicative of ER stress.

Differential alterations in the expression and activity of matrix metalloproteinases 2 and 9 after transient cerebral ischemia in mice.

Abnormal expression and activity of matrix metalloproteinases (MMPs) may contribute to the pathophysiology of cerebral disease such as ischemic injury. In this study, we compared the cellular localization, expression, and activity of MMP-2 and -9 in relation to the evolution of neuronal damage 24 and 72 h after transient global ischemia. In response to ischemia, there was a generalized increase in cellular MMP-2 immunoreactivity at 24-h reperfusion (in neurons, glia and vessels) whereas at 72-h reperfusion the increase in MMP-2 was predominantly in glia. These glial alterations contributed to a significant increase in pro MMP-2 levels in ischemic regions (P < 0.01) as measured by zymography. In contrast, MMP-9 was predominantly upregulated in neurons and this was significantly different to shams at 24- and 72-h reperfusion after ischemia (P < 0.05). Notably, a dramatic increase in proteolytic activity in neurons was observed 24 h after ischemia and this response was absent at 72 h post-ischemia. The present data are supportive of a role for MMPs in contributing to neuronal injury after ischemia.