High Performance Proteomics: 7th HUPO Brain Proteome Project Workshop March 7-9, 2007 Wellcome Trust Conference Centre, Hinxton, UK

The Wellcome Trust Conference Centre at Hinxton, UK, was the meeting place of the 7th HUPO Brain Proteome Project Workshop entitled "High Performance Proteomics". It started on Wednesday, March 7, 2007 with a steering committee meeting followed by a two days series of talks dealing with the standardization and handling of tissues, body fluids as well as of proteomics data. The presentation and accompanying vivid discussions created a picture of actual strategies and standards in recent proteomics.     

Pre-mRNA secondary structures influence exon recognition

The secondary structure of a pre-mRNA influences a number of processing steps including alternative splicing. Since most splicing regulatory proteins bind to single-stranded RNA, the sequestration of RNA into double strands could prevent their binding. Here, we analyzed the secondary structure context of experimentally determined splicing enhancer and silencer motifs in their natural pre-mRNA context. We found that these splicing motifs are significantly more single-stranded than controls. These findings were validated by transfection experiments, where the effect of enhancer or silencer motifs on exon skipping was much more pronounced in single-stranded conformation. We also found that the structural context of predicted splicing motifs is under selection, suggesting a general importance of secondary structures on splicing and adding another level of evolutionary constraints on pre-mRNAs. Our results explain the action of mutations that affect splicing and indicate that the structural context of splicing motifs is part of the mRNA splicing code.     

Expression level and agonist-binding affect the turnover, ubiquitination and complex formation of peroxisome proliferator activated receptor beta

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that modulate target gene expression in response to fatty acid ligands. Their regulation by post-translational modifications has been reported but is poorly understood. In the present study, we investigated whether ligand binding affects the turnover and ubiquitination of the PPARbeta subtype (also known as PPARdelta). Our data show that the ubiquitination and degradation of PPARbeta is not significantly influenced by the synthetic agonist GW501516 under conditions of moderate PPARbeta expression. By contrast, the overexpression of PPARbeta dramatically enhanced its degradation concomitant with its polyubiquitination and the formation of high molecular mass complexes containing multiple, presumably oligomerized PPARbeta molecules that lacked stoichiometical amounts of the obligatory PPARbeta dimerization partner, retinoid X receptor. The formation of these apparently aberrant complexes, as well as the ubiquitination and destabilization of PPARbeta, were strongly inhibited by GW501516. Our findings suggest that PPARbeta is subject to complex post-translational regulatory mechanisms that partly may serve to safeguard the cell against deregulated PPARbeta expression. Furthermore, our data have important implications regarding the widespread use of overexpression systems to evaluate the function and regulation of PPARs.     

Siderophores drive invasion dynamics in bacterial communities through their dual role as public good versus public bad

Microbial invasions can compromise ecosystem services and spur dysbiosis and disease in hosts. Nevertheless, the mechanisms determining invasion outcomes often remain unclear. Here, we examine the role of iron-scavenging siderophores in driving invasions of Pseudomonas aeruginosa into resident communities of environmental pseudomonads. Siderophores can be ‘public goods’ by delivering iron to individuals possessing matching receptors; but they can also be ‘public bads’ by withholding iron from competitors lacking these receptors. Accordingly, siderophores should either promote or impede invasion, depending on their effects on invader and resident growth. Using supernatant feeding and invasion assays, we show that invasion success indeed increased when the invader could use its siderophores to inhibit (public bad) rather than stimulate (public good) resident growth. Conversely, invasion success decreased the more the invader was inhibited by the residents’ siderophores. Our findings identify siderophores as a major driver of invasion dynamics in bacterial communities under iron-limited conditions.     

Individual‐ versus group‐optimality in the production of secreted bacterial compounds

How unicellular organisms optimize the production of compounds is a fundamental biological question. While it is typically thought that production is optimized at the individual‐cell level, secreted compounds could also allow for optimization at the group level, leading to a division of labor where a subset of cells produces and shares the compound with everyone. Using mathematical modeling, we show that the evolution of such division of labor depends on the cost function of compound production. Specifically, for any trait with saturating benefits, linear costs promote the evolution of uniform production levels across cells. Conversely, production costs that diminish with higher output levels favor the evolution of specialization–especially when compound shareability is high. When experimentally testing these predictions with pyoverdine, a secreted iron‐scavenging compound produced by Pseudomonas aeruginosa, we found linear costs and, consistent with our model, detected uniform pyoverdine production levels across cells. We conclude that for shared compounds with saturating benefits, the evolution of division of labor is facilitated by a diminishing cost function. More generally, we note that shifts in the level of selection from individuals to groups do not solely require cooperation, but critically depend on mechanistic factors, including the distribution of compound synthesis costs.     

Pertussis toxin-sensitive G-proteins inhibit fibroblast growth factor-induced signaling in pancreatic acini

Signal transduction of fibroblast growth factor (FGF) receptors is known to involve tyrosine phosphorylation of several substrates, including Grb2, phospholipase C-γ, and phosphatidylinositol 3-kinase, whereas the role of G-proteins in FGF receptor signaling is controversial. In the present study we investigated the role of G-proteins in FGF receptor signaling in rat pancreatic acini. Immunological analysis revealed the presence of FGF receptor and phospholipase C-γ1 in rat pancreatic acini. Both basic fibroblast growth factor (FGF-2) and guanosine 5′-(γ-O-thio)triphosphate (GTPγS) caused an increase in inositol 1,4,5-trisphosphate (1,4,5-IP₃) production and amylase release. Combined stimulation of the acini with GTPγS and FGF-2 led to a decrease of these responses as compared to the effect of the single substances. When pancreatic acini were preincubated with FGF-2 (1 nM) or vehicle (water) ADP-ribosylation of the α-subunit of G_i-type G-proteins by pertussis toxin was reduced in membranes prepared from FGF-2 pretreated acini as compared to control acini, suggesting functional interaction of FGF receptors with G_i-proteins. Pretreatment of acini with pertussis toxin which inhibits G_i-type G-proteins abolished the inhibitory effect of GTPγS on FGF-induced 1,4,5-IP₃ production and amylase release, whereas the stimulatory effects of FGF-2 and GTPγS on these parameters remained unchanged. In conclusion, these results show communication of FGF receptors and G_i-type G-proteins and that G_i-type G-proteins exert an inhibitory influence on FGF-induced activation of phosphoinositide-specific phospholipase C in pancreatic acinar cells. © 1996 Wiley-Liss, Inc.     

Molecular,Viral and Clinical Features of Alcohol- and Non-Alcohol-Induced Liver Injury

Hepatic cells are sensitive to internal and external signals. Ethanol is one of the oldest and most widely used drugs in the world. The focus on the mechanistic engine of the alcohol-induced injury has been in the liver, which is responsible for the pathways of alcohol metabolism. Ethanol undergoes a phase I type of reaction, mainly catalyzed by the cytoplasmic enzyme, alcohol dehydrogenase (ADH), and by the microsomal ethanol-oxidizing system (MEOS). Reactive oxygen species (ROS) generated by cytochrome (CYP) 2E1 activity and MEOS contribute to ethanol-induced toxicity. We aimed to: (1) Describe the cellular, pathophysiological and clinical effects of alcohol misuse on the liver; (2) Select the biomarkers and analytical methods utilized by the clinical laboratory to assess alcohol exposure; (3) Provide therapeutic ideas to prevent/reduce alcohol-induced liver injury; (4) Provide up-to-date knowledge regarding the Corona virus and its affect on the liver; (5) Link rare diseases with alcohol consumption. The current review contributes to risk identification of patients with alcoholic, as well as non-alcoholic, liver disease and metabolic syndrome. Additional prevalence of ethnic, genetic, and viral vulnerabilities are presented.     

Subtype-selectivity of metal-dependent methionine aminopeptidase inhibitors

Inhibitors of methionine aminopeptidases (MetAPs) are treatment options for various pathological conditions. Several inhibitor classes have been described previously, but only few data on the subtype selectivity, which is of crucial importance for these enzymes, is available. We present a systematic study on the subtype- and species-selectivity of MetAP inhibitors that require the binding of an auxiliary metal ion. This includes, in particular, compounds based on the benzimidazole pharmacophore, but also hydroxyquinoline and picolinic acid derivatives. Our data indicates that a significant degree of selectivity can be attained with metal-dependent MetAP inhibitors.     

Identification of a cis-acting element and a novel trans-acting factor of the glutamine synthetase gene in liver cells

In the mammalian liver the expression of the enzyme glutamine synthetase (GS) is restricted to a small population of hepatocytes. In cells expressing the enzyme up to 3.5% of total cellular protein is GS. In order to identify enhancer elements contributing to this extraordinarily high level of expression we focused on a region roughly 2.5 kbp upstream of the GS promoter. Gel mobility shift assays revealed binding of an unknown protein within the most distal part of this region and reportergene assays demonstrated that roughly 60 bp downstream from position -2503 are indispensable for protein binding and the full effect of the enhancer. In UV cross-link analysis a 38 kDa nuclear protein that binds to the sequence was identified in rat hepatocytes. This nuclear protein, designated as upstream binding factor of the GS gene (UFGS) seems to play an important role in high-level expression of GS in liver.     

A novel class of oxylipins, sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl Diglyceride, from Arabidopsis thaliana

The cyclic derivative of 13(S)-hydroperoxolinolenic acid, 12-oxophytodienoic acid, serves as a signal transducer in higher plants, mediating mechanotransductory processes and plant defenses against a variety of pathogens, and also serves as a precursor for the biosynthesis of jasmonic acid, a mediator of plant herbivore defense. Biosynthesis of 12-oxophytodienoic acid from alpha-linolenic acid occurs in plastids, mainly in chloroplasts, and is thought to start with free linolenic acid liberated from membrane lipids by lipase action. In Arabidopsis thaliana, the glycerolipid fraction contains esterified 12-oxophytodienoic acid, which can be released enzymatically by sn1-specific, but not by sn2-specific, lipases. The 12-oxophytodienoyl glycerolipid fraction was isolated, purified, and characterized. Enzymatic, mass spectrometric, and NMR spectroscopic data allowed us to establish the structure of the novel oxylipin as sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl diglyceride. The novel class of lipids is localized in plastids. Purified monogalactosyl diglyceride was not converted to the sn1-(12-oxophytodienoyl) derivative by the combined action of (soybean) lipoxygenase and (A. thaliana) allene oxide synthase, an enzyme ensemble that converts free alpha-linolenic acid to free 12-oxophytodienoic acid. When leaves were wounded, a significant and transient increase in the level of (12-oxophytodienoyl)-monogalactosyl diglyceride was observed. In A. thaliana, the major fraction of 12-oxophytodienoic acid occurs esterified at the sn1 position of the plastid-specific glycerolipid, monogalactosyl diglyceride.