Phosducin-like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins

Retinal phosducin is known to sequester transducin Gbetagamma, thereby modulating transducin activity. Phosducin is a member of a family of phosducin-like proteins (PhLP) found in eukaryotes. Phylogeny of 33 phosducin-like proteins from metazoa, plants and lower eukaryotes identified three distinct groups named phosducin-I-III. We discovered three phlp genes in Dictyostelium, each encoding a phosducin-like protein of a different group. Disruption of the phlp1 gene strongly impaired G-protein signalling, apparently due to mislocalization of Gbetagamma in phlp1-null cells. GFP-Gbeta and GFP-Ggamma are membrane associated in wild-type cells, but cytosolic in phlp1-null cells. Phlp2 disruption is lethal due to a synchronous collapse of the cells after 16-17 cell divisions. Phlp3 disruptants show no abnormal phenotype. These results establish a role for phosducin-like proteins in facilitating folding, localization or function of proteins, in addition to modulating G-protein signalling.     

A proteomics approach to understanding protein ubiquitination

There is a growing need for techniques that can identify and characterize protein modifications on a large or global scale. We report here a proteomics approach to enrich, recover, and identify ubiquitin conjugates from Saccharomyces cerevisiae lysate. Ubiquitin conjugates from a strain expressing 6xHis-tagged ubiquitin were isolated, proteolyzed with trypsin and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for amino acid sequence determination. We identified 1,075 proteins from the sample. In addition, we detected 110 precise ubiquitination sites present in 72 ubiquitin-protein conjugates. Finally, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo. The methodology described here provides a general tool for the large-scale analysis and characterization of protein ubiquitination.     

A panel of candidate tumor antigens in colorectal cancer revealed by the serological selection of a phage displayed cDNA expression library

In the last few years it has been shown that the humoral immune response in cancer patients is a rich source of putative cancer vaccine candidates. To fully explore the complex information present within the Ab repertoire of cancer patients, we have applied a method, serological Ag selection, to molecularly define tumor Ags recognized by the humoral immune response in colorectal cancer (CRC). First, we built a cDNA display library by cloning a cDNA library from CRC cell line HT-29 for expression as a fusion protein with a filamentous phage minor coat protein, pVI. This cDNA display library was then enriched on pooled sera from CRC patients who had undergone active specific immunization with autologous tumor. We identified a panel of 19 clones reactive with the serum pool. Seventeen of 19 (89%) clones showed reactivity with one or more of the eight Ag-reactive sera, conversely six of eight (75%) sera were reactive with at least one of the 19 clones. Sequencing revealed that these 19 clones represented 13 different Ags. A detailed serological analysis of the 13 different Ags showed preferential reactivity to sera of cancer patients for six different Ags. Four of these Ags displayed increased serum reactivity after the active specific immunization procedure. Furthermore, one of the six Ags, a novel Ag homologous to HSPC218, showed restricted expression in normal testis, suggesting that it belongs to the cancer-testis Ag family. Some of the Ags we have identified may be candidates for tumor vaccination, for sero-diagnosis of cancer, as prognostic markers, or as probes for monitoring tumor cell-based vaccination trials.     

TCR-like human antibodies expressed on human CTLs mediate antibody affinity-dependent cytolytic activity

The permanent genetic programming via gene transfer of autologous T cells with cell surface receptors directed toward tumor-related Ags holds great promise for the development of more-specific tumor therapies. In this study we have explored the use of Abs directed to MHC-peptide complexes (or TCR-like Abs) to engraft CTLs with exquisite specificity for cancer cells. First, we affinity matured in vitro a previously selected TCR-like Ab, Fab-G8, which is highly specific for the peptide melanoma-associated Ag-A1 presented by the HLA-A1 molecule. A combination of L chain shuffling, H chain-targeted mutagenesis, and in vitro selection of phage display libraries yielded a Fab-G8 Ab derivative, Fab-Hyb3, with an 18-fold improved affinity yet identical peptide fine specificity. Fab-G8 and Fab-Hyb3 were expressed on primary human T lymphocytes as cell surface-anchored Fab, demonstrating that T cells expressing the high-affinity Fab-Hyb3 molecule eradicate tumor cells much more effectively. Furthermore, the gain in ligand-binding affinity resulted in a 2-log improvement in the detection of peptide/MHC complexes on melanoma-associated Ag-A1 peptide-loaded cells. In summary, an affinity-matured Ab specifically recognizing a cancer-related peptide/MHC complex was generated and used to improve the tumor cell killing capacity of human T cells. This strategy, based on engraftment of T cells with in vitro engineered Abs, is an attractive alternative to the laborious, and in many cases unsuccessful, generation of highly potent tumor-specific T lymphocytes.     

Differential effects of ammonium and nitrate on three heathland species

Arnica montana and Cirsium dissectum, typical of species-rich heathlands and acidic grasslands, have declined rapidly in The Netherlands in recent years. Field surveys suggest that the decline is caused by soil acidification as a result of enhanced atmospheric N and S deposition. Therefore, the survival, growth and development of these species were studied in a water culture experiment, using nutrient solutions which differed both in mineral nitrogen form and in ammonium concentration. For comparison, the performance of a third, acid tolerant species, Calluna vulgaris, was studied. The results showed that both Arnica and Cirsium performed better using nitrate than when using ammonium as a sole nitrogen source, whereas ammonium toxicity became apparent when ammonium concentrations were raised above 100 µM. Ammonium toxicity was expressed by an increase in mortality of Arnica plants with increasing ammonium concentrations and by a reduction of biomass in Arnica and Cirsium. Furthermore, cation concentrations in both roots and shoots decreased when ammonium was supplied as a nitrogen source. In contrast, Calluna showed optimal development when using ammonium as a sole nitrogen source. In this species, only root biomass was negatively affected by high ammonium concentrations. The ecological implications of these preferences are discussed in relation to soil acidification.     

Stimulation of sex pheromone production by proteinaceous extracts of the bursa copulatrix in the redbanded leafroller moth

Pheromone biosynthesis in the redbanded leafroller moth, Argyrotaenia velutinana, was stimulated by homogenates of the bursa copulatrix. Although pheromonotropic activity was also extractable from the ovary, the activity of pheromone biosynthesis activating neuropeptide (PBAN) or bursa extracts was not impaired in isolated abdomens by removal of the ovary. Response to the bursa extracts was dependent on the dose administered and the time of incubation. Amounts of pheromone present in adult females of different ages appeared to be correlated with the extractable amount of pheromonotropic activity from their bursa copulatrix. Decapitation did not result in the suppression of burse factor production. Homogenates of the bursa elicited similar effects in both isolated gland and isolated abdomen incubations, but the brain neuropeptide, PBAN, was less active in the former than in the latter. Bursa extracts stimulated pheromone production in isolated abdomen incubations deprived of the bursa copulatrix, but PBAN did not. Loss of activity of bursa homogenates after treatment with either pronase E or carboxypeptidase Y indicated that the pheromonotropic factor is a proteinaceous substance. The mechanism through which pheromone production is regulated in redbanded leafroller moths is discussed. © 1992 Wiley-Liss, Inc.     

Characterizing the Hez-PBAN gene products in neuronal clusters with immunocytochemistry and MALDI MS

Methods to characterize pheromone biosynthesis activating neuropeptide (PBAN) and other PBAN gene encoded neuropeptides (PGN) from individual subesophageal ganglion neuronal clusters of the corn earworm moth, Helicoverpa zea, were developed. Individual antisera against the N-terminal sequence to PBAN and each of the three PGNs from the Hez-PBAN prohormone were developed, and their specificity determined. In all cases, each antiserum stains the same three groups of subesophageal ganglion ventral midline neurons-the mandibular, maxillary and labial neurons-in both adult females and males. These results were confirmed using matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) of individual subesophageal ganglion neuronal clusters. Using mass spectrometry, the amidated PGN-24 was not detected but an N-terminally extended form is observed that is two amino acids longer. Other peptides resulting from the processing of the Hez-PBAN prohormone were detected. Using both the specific antisera and the cellular profiling abilities of MALDI MS, the roles of individual members of the Hez-PBAN prohormone derived peptides can now be explored.