Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database

HUPO initiated the Plasma Proteome Project (PPP) in 2002. Its pilot phase has (1) evaluated advantages and limitations of many depletion, fractionation, and MS technology platforms; (2) compared PPP reference specimens of human serum and EDTA, heparin, and citrate-anti-coagulated plasma; and (3) created a publicly-available knowledge base (www.bioinformatics.med.umich.edu/hupo/ppp; www.ebi.ac.uk/pride). Thirty-five participating laboratories in 13 countries submitted datasets. Working groups addressed (a) specimen stability and protein concentrations; (b) protein identifications from 18 MS/MS datasets; (c) independent analyses from raw MS-MS spectra; (d) search engine performance, subproteome analyses, and biological insights; (e) antibody arrays; and (f) direct MS/SELDI analyses. MS-MS datasets had 15 710 different International Protein Index (IPI) protein IDs; our integration algorithm applied to multiple matches of peptide sequences yielded 9504 IPI proteins identified with one or more peptides and 3020 proteins identified with two or more peptides (the Core Dataset). These proteins have been characterized with Gene Ontology, InterPro, Novartis Atlas, OMIM, and immunoassay-based concentration determinations. The database permits examination of many other subsets, such as 1274 proteins identified with three or more peptides. Reverse protein to DNA matching identified proteins for 118 previously unidentified ORFs. We recommend use of plasma instead of serum, with EDTA (or citrate) for anticoagulation. To improve resolution, sensitivity and reproducibility of peptide identifications and protein matches, we recommend combinations of depletion, fractionation, and MS/MS technologies, with explicit criteria for evaluation of spectra, use of search algorithms, and integration of homologous protein matches. This Special Issue of PROTEOMICS presents papers integral to the collaborative analysis plus many reports of supplementary work on various aspects of the PPP workplan. These PPP results on complexity, dynamic range, incomplete sampling, false-positive matches, and integration of diverse datasets for plasma and serum proteins lay a foundation for development and validation of circulating protein biomarkers in health and disease.     

A generic system for the Escherichia coli cell-surface display of lipolytic enzymes

EstA is an outer membrane-anchored esterase from Pseudomonas aeruginosa. An inactive EstA variant was used as an anchoring motif for the Escherichia coli cell-surface display of lipolytic enzymes. Flow cytometry analysis and measurement of lipase activity revealed that Bacillus subtilis lipase LipA, Fusarium solani pisi cutinase and one of the largest lipases presently known, namely Serratia marcescens lipase were all efficiently exported by the EstA autotransporter and also retained their lipolytic activities upon cell surface exposition. EstA provides a useful tool for surface display of lipases including variant libraries generated by directed evolution thereby enabling the identification of novel enzymes with interesting biological and biotechnological ramifications.     

Co-expression of nicastrin and presenilin rescues a loss of function mutant of APH-1

gamma-Secretase is an intramembrane-cleaving aspartyl protease complex that mediates the final cleavage of beta-amyloid precursor protein to liberate the neurotoxic amyloid-beta peptide implicated in Alzheimer's disease. The four proteins presenilin (PS), nicastrin (NCT), APH-1, and PEN-2 are sufficient to reconstitute gamma-secretase activity in yeast. Although PS seems to contribute the catalytic core of the gamma-secretase complex, no distinct function could be attributed to the other components so far. In Caenorhabditis elegans, mutation of a glycine to an aspartic acid within a conserved GXXXG motif in the fourth transmembrane domain of APH-1 causes a loss of function phenotype. Surprisingly, we now found that the human homologue APH-1a carrying the equivalent mutation G122D is fully active in yeast co-expressing PS1, NCT, and PEN-2. To address this discrepancy, we expressed APH-1a G122D in HEK293 cells. As reported previously, overexpressed APH-1a G122D was not incorporated into the gamma-secretase complex. Separate overexpression of PS1, NCT, or PEN-2 together with APH-1a G122D allowed the formation of heterodimers lacking the other endogenous components. Only the combined overexpression of PS1 and NCT together with APH-1a G122D facilitated the formation of a fully active gamma-secretase complex. Under these conditions, APH-1a G122D supported the production of normal amounts of Abeta. We conclude that cooperative effects may stabilize a trim-eric complex of APH-1a G122D together with PS1 and NCT. Upon successful complex assembly, the GXXXG motif becomes dispensable for gamma-secretase activity.     

Automated counting of mammalian cell colonies by means of a flat bed scanner and image processing.

BACKGROUND: Clonogenic assays are used frequently to measure the cell killing and mutagenic effects of radiation and other agents. Clonogenic assays carried out manually are tedious and time-consuming and involve a significant element of subjectivity. However, several commercial automatic colony counters are available. Based on CCD video imaging and image analysis they are relatively expensive and can analyze only one petri dish at a time. METHOD: We have developed a cheaper and more efficient device, which employs a flat bed scanner to image 12 60-mm petri dishes at a time. Two major problems in automated colony counting are the clustering of colonies and edge effects. By using standard image analysis and implementing an inflection point algorithm, these problems were greatly diminished. The resulting system was compared with two manual colony counts, as well as with automated counts with the Oxford Optronix ColCount colony counter for cell lines V79 and HaCaT. RESULTS: Comparisons assuming the manual counts to be correct showed that our automatic counter was slightly more accurate than the commercial unit. CONCLUSIONS: As a whole, our automated colony counter performed significantly better than the commercial unit with regard to processing time, cost and accuracy.     

Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy

Single-molecule force-spectroscopy was employed to unfold and refold single sodium-proton antiporters (NhaA) of Escherichia coli from membrane patches. Although transmembrane alpha-helices and extracellular polypeptide loops exhibited sufficient stability to individually establish potential barriers against unfolding, two helices predominantly unfolded pairwise, thereby acting as one structural unit. Many of the potential barriers were detected unfolding NhaA either from the C-terminal or the N-terminal end. It was found that some molecular interactions stabilizing secondary structural elements were directional, while others were not. Additionally, some interactions appeared to occur between the secondary structural elements. After unfolding ten of the 12 helices, the extracted polypeptide was allowed to refold back into the membrane. After five seconds, the refolded polypeptide established all secondary structure elements of the native protein. One helical pair showed a characteristic spring like snap in into its folded conformation, while the refolding process of other helices was not detected in particular. Additionally, individual helices required characteristic periods of time to fold. Correlating these results with the primary structure of NhaA allowed us to obtain the first insights into how potential barriers establish and determine the folding kinetics of the secondary structure elements.     

Modafinil improves performance in the multiple T-Maze and modifies GluR1, GluR2, D2 and NR1 receptor complex levels in the C57BL/6J mouse

Modafinil has been shown to modify behavioural and cognitive functions and to effect several brain receptors. Effects, however, were not observed at the receptor protein complex level and it was therefore the aim of the study to train mice in the multiple T-Maze (MTM) as a paradigm for spatial memory and to determine paralleling brain receptor complex levels. Sixty C57BL/6J mice were used in the study and divided into four groups (trained drug injected; trained vehicle injected; yoked drug injected; yoked vehicle injected). Animals obtained training for 4?days and were killed 6?h following the last training session on day 4. Hippocampi were dissected from the brain, membrane fractions were prepared by ultracentrifugation and were run on blue-native gels and immunoblotted with antibodies against major brain receptors. Modafinil treatment led to decreased latency and increased average speed, but not to changes in pathlength and number of correct decisions in the MTM. Drug effects were modifying receptor complexes of GluR1, GluR2, D2 and NR1. Training effects on receptor complex levels were observed for GluR3, D1 and nicotinic acetylcholine receptor alpha 7 (Nic7). GluR1 levels were correlating with GluR2 and D1 levels were correlating with D2 and NR1. Involvement of the glutamatergic, NMDA, dopaminergic and nicotinergic system in modafinil and memory training were herein described for the first time. A brain receptor complex pattern was revealed showing the concerted action following modafinil treatment.     

A TNF receptor 2 selective agonist rescues human neurons from oxidative stress-induced cell death

Tumor necrosis factor (TNF) plays a dual role in neurodegenerative diseases. Whereas TNF receptor (TNFR) 1 is predominantly associated with neurodegeneration, TNFR2 is involved in tissue regeneration and neuroprotection. Accordingly, the availability of TNFR2-selective agonists could allow the development of new therapeutic treatments of neurodegenerative diseases. We constructed a soluble, human TNFR2 agonist (TNC-scTNF(R2)) by genetic fusion of the trimerization domain of tenascin C to a TNFR2-selective single-chain TNF molecule, which is comprised of three TNF domains connected by short peptide linkers. TNC-scTNF(R2) specifically activated TNFR2 and possessed membrane-TNF mimetic activity, resulting in TNFR2 signaling complex formation and activation of downstream signaling pathways. Protection from neurodegeneration was assessed using the human dopaminergic neuronal cell line LUHMES. First we show that TNC-scTNF(R2) interfered with cell death pathways subsequent to H(2)O(2) exposure. Protection from cell death was dependent on TNFR2 activation of the PI3K-PKB/Akt pathway, evident from restoration of H(2)O(2) sensitivity in the presence of PI3K inhibitor LY294002. Second, in an in vitro model of Parkinson disease, TNC-scTNF(R2) rescues neurons after induction of cell death by 6-OHDA. Since TNFR2 is not only promoting anti-apoptotic responses but also plays an important role in tissue regeneration, activation of TNFR2 signaling by TNC-scTNF(R2) appears a promising strategy to ameliorate neurodegenerative processes.     

Effectiveness of honey bees in delivering the biocontrol agent Bacillus subtilis to blueberry flowers to suppress mummy berry disease

Honey bees are important pollinators of commercial blueberries in the southeastern United States, and blueberry producers often use supplemental bees to achieve adequate fruit set. However, honey bees also vector the plant pathogenic fungus Monilinia vaccinii-corymbosi which infects open blueberry flowers through the gynoecial pathway causing mummy berry disease. Here, we report the results of a 3-year field study to test the hypothesis that using bee hives equipped with dispensers containing the biocontrol product Serenade, a commercial formulation of the bacterium Bacillus subtilis which has shown activity against flower infection by M. vaccinii-corymbosi in laboratory experiments, can reduce mummy berry disease incidence when honey bees are used as pollinators in blueberries. Individual honey bees carried 5.1–6.4 × 10⁵ colony-forming units (CFU) of B. subtilis when exiting hive-mounted dispensers with Serenade. On caged rabbiteye blueberry bushes in the field, population densities of B. subtilis vectored by honey bees reached a carrying capacity of <10³ CFU per flower stigma within 2 days of exposure, and there was a highly significant non-linear relationship between B. subtilis populations per stigma and bee activity, expressed as number of legitimate flower visits per time interval per cage (R = 0.6928, P < 0.0001, n = 32). Honey bee density (1600 or 6400 individuals per 5.8-m³ cage) and Serenade treatment (presence or absence of the product in hive-mounted dispensers) significantly (P < 0.05) affected the incidence of fruit mummification on caged bushes, whereby increasing bee density increased disease incidence and application of Serenade reduced disease levels. Taken together, results of this study suggest that use of a hive-dispersed biocontrol product such as Serenade as a supplement during pollination can reduce the risk of mummy berry disease. This may be a prudent practice that optimizes the benefits to pollination of high bee densities while reducing the associated disease-vectoring risk.