Report from the Wellcome Trust/EBA “Perspectives in Clinical Proteomics” retreat--a strategy to implement next-generation proteomic analyses to the clinic for patient benefit: pathway to translation

Proteomics has long been thought to hold the promise of producing results of clinical utility which will influence patient treatment and outcomes. A recent Wellcome Trust/EBI meeting and retreat--“Perspectives in Clinical Proteomics”--brought together experts from a broad range of stakeholder groups with an interest in ensuring proteomics achieves this aim. This viewpoint presents views derived from these forums, proposing a pathway for the development of next-generation proteomic analyses in the clinical setting from selection of candidates through to their validation and ultimate demonstration of utility through health technology assessments. Although not meant to be all encompassing, important elements for proteomics researchers to consider are presented.     

Mitochondria: A mirror into cellular dysfunction in heart disease

Cardiovascular (CV) disease is the single most significant cause of morbidity and mortality worldwide. The emerging global impact of CV disease means that the goals of early diagnosis and a wider range of treatment options are now increasingly pertinent. As such, there is a greater need to understand the molecular mechanisms involved and potential targets for intervention. Mitochondrial function is important for physiological maintenance of the cell, and when this function is altered, the cell can begin to suffer. Given the broad range and significant impacts of the cellular processes regulated by the mitochondria, it becomes important to understand the roles of the proteins associated with this organelle. Proteomic investigations of the mitochondria are hampered by the intrinsic properties of the organelle, including hydrophobic mitochondrial membranes; high proportion of basic proteins (pI greater than 8.0); and the relative dynamic range issues of the mitochondria. For these reasons, many proteomic studies investigate the mitochondria as a discrete subproteome. Once this has been achieved, the alterations that result in functional changes with CV disease can be observed. Those alterations that lead to changes in mitochondrial function, signaling and morphology, which have significant implications for the cardiomyocyte in the development of CV disease, are discussed.     

Optimal modification of annexin V with fluorescent dyes

The many uses of chemically modified annexin Vs necessitate an understanding of the optimal degree of modification and modification sites of the protein. When reacted with the N-hydroxysuccinimide ester of Cy5.5, annexin V with one modification per mole of protein retained its affinity for phosphatidylserine of apoptotic cells, whereas modification with two dyes per mole of protein caused a complete loss of activity. A tryptic digest LC/MS method was used to identify the modification sites as either of two closely spaced lysine residues, in position 286 or 290. The crystal structure indicated the location of these lysines was distal to the phosphatidylserine binding sites on annexin V. These results can be used to develop active or inactive fluorescent control annexin V proteins and to suggest strategies for attaining higher levels of modification with retention of bioactivity.     

Quantitative measurement of atomic sodium in the plume of a single burning coal particle

The release of volatile sodium during coal combustion is a significant factor in the fouling and corrosion of heat transfer surfaces within industrial coal-fired boilers. A method for measuring the temporal release of atomic sodium from a single coal particle is described. Laser absorption was used to calibrate laser-induced fluorescence measurements of atomic sodium utilising the sodium D1 line (589.59 nm) in a purpose-designed flat flame environment. The calibration was then applied to planar laser-induced fluorescence measurements of sodium atoms in the plume from a single Victorian brown coal particle (53 mg) suspended within the flat flame. The peak concentration of atomic sodium was approximately 64.1 ppb after 1080.5 s, which appears to correspond to the end of char combustion. To our knowledge this is the first in situ quantitative measurement of the concentration field of atomic sodium in the plume above a burning particle. A simple kinetic model has been used to estimate the rate of sodium decay in the post-flame gases. Comparison of the estimated and measured decay rates showed reasonable agreement.     

Activation of CPP32-like proteases is not sufficient to trigger apoptosis: inhibition of apoptosis by agents that suppress activation of AP24, but not CPP32-like activity

The 24-kD apoptotic protease (AP24) is a serine protease that is activated during apoptosis and has the capacity to activate internucleosomal DNA fragmentation in isolated nuclei. This study examined the following: (a) the functional relationship between AP24 and the CPP32-like proteases of the caspase family; and (b) whether activation of CPP32-like proteases is sufficient to commit irreversibly a cell to apoptotic death. In three different leukemia cell lines, we showed that agents that directly (carbobenzoxy-Ala-Ala-borophe (DK120) or indirectly inhibit activation of AP24 (protein kinase inhibitors, basic fibroblast growth factor, tosylphenylalaninechloromethylketone, and caspase inhibitors) protected cells from apoptosis induced by TNF or UV light. Only the caspase inhibitors, however, prevented activation of CPP32-like activity as revealed by cleavage of the synthetic substrate, DEVD-pNa, by cell cytosols, and also by in vivo cleavage of poly (ADP-ribosyl) polymerase, a known substrate of CPP32. Activation of DEVD-pNa cleaving activity without apoptosis was also demonstrated in two variants derived from the U937 monocytic leukemia in the absence of exogenous inhibitors. Cell-permeable peptide inhibitors selective for CPP32-like proteases suppressed AP24 activation and apoptotic death. These findings indicate that CPP32-like activity is one of several upstream signals required for AP24 activation. Furthermore, activation of CPP32-like proteases alone is not sufficient to commit irreversibly a cell to apoptotic death under conditions where activation of AP24 is inhibited.     

The role of O-GlcNAc signaling in the pathogenesis of diabetic retinopathy

Diabetic retinopathy is a leading cause of blindness worldwide. Despite laser and surgical treatments, antiangiogenic and other therapies, and strict metabolic control, many patients progress to visual impairment and blindness. New insights are needed into the pathophysiology of diabetic retinopathy in order to develop new methods to improve the detection and treatment of disease and the prevention of blindness. Hyperglycemia and diabetes result in increased flux through the hexosamine biosynthetic pathway, which, in turn, results in increased PTM of Ser/Thr residues of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation is involved in regulation of many nuclear and cytoplasmic proteins in a manner similar to protein phosphorylation. Altered O-GlcNAc signaling has been implicated in the pathogenesis of diabetes and may play an important role in the pathogenesis of diabetic retinopathy. The goal of this review is to summarize the biology of the hexosamine biosynthesis pathway and O-GlcNAc signaling, to present the current evidence for the role of O-GlcNAc signaling in diabetes and diabetic retinopathy, and to discuss future directions for research on O-GlcNAc in the pathogenesis of diabetic retinopathy.     

Slope deposits and water paths in a spring catchment, Frankenwald, Bavaria, Germany

The spring catchment under study is underlain by shale, on which several layers of slope sediments (cover-beds) are deposited. The upper of these layers contain eolian fines mixed into shale-derived debris, which latter material entirely comprises the basal cover-bed. Due to its dislocation by solifluction, the shale debris has a tegular structure. This leads to hydraulic anisotropy, particularly where no fine earth occurs. Thus, water that seeps into such material is forced to flow laterally while vertical movement is impeded. The basal cover-bed therefore hosts a deep aquifer. Only during major discharge events, excess hydrostatic pressure makes water flow into upper parts of the soils, being mixed with surficially interflowing water. Most of the time, however, there are two levels of water flow at different depths with different dwelling times, which finding is supported by probably geogenic sulphur isotopes in the deeply flowing water that are different from precipitation water.KurzfassungDas bearbeitete Quelleinzugsgebiet liegt in Tonschiefer, welcher mit Deckschichten bedeckt ist. In die oberen Deckschichten wurde Löß eingearbeitet, während die untere ausschließlich aus umgelagertem Anstehenden besteht. Dieses Material wurde solifluidal verlagert und dabei mit einer dachziegelartigen Struktur abgelagert. Diese führt zu hydraulischer Anisotropie besonders in feinerdearmen Schichten. Wasser, das darin einsickert, fließt bevorzugt lateral, während vertikale Bewegungen stark behindert werden. Deshalb ist in der Basislage ein tieferes Aquifer entstanden. Während ausgeprägter Abflußereignisse wird der Überdruck darin jedoch so groß, daß es zu einem Aufpressen des Wasser in hangende Schichten und dort zur Vermischung mit höherem Zwischenabfluß kommt. Zumeist aber gibt es zwei unterschiedliche Interflow-Stockwerke in verschiedenen Tiefen. Dies wird bestätigt durch eine wahrscheinlich geogene Sulfatbeimischung im tieferen Stockwerk, die sich vom Niederschlagswasser in den Isotopen unterscheidet.     

Construction and optimization of a CC49-based scFv-beta-lactamase fusion protein for ADEPT

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).