Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome

The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.     

Proteome analysis of chemically induced mouse liver tumors with different genotype

Mouse liver tumors frequently harbor mutations in Ha-ras, B-raf, or Ctnnb1 (encoding beta-catenin). We conducted a proteome analysis with protein extracts from normal mouse liver and from liver tumors which were induced by a single injection of N-nitrosodiethylamine (DEN) as initiator followed by multiple injections of two different polychlorinated biphenyls (PCBs) as tumor promoters, or corn oil as a control. Liver tumors were stratified into two classes: they were either mutated in Ctnnb1 and positive for the marker glutamine synthetase (GS(+)), or they lacked Ctnnb1 mutations and were therefore GS-negative (GS(-)). Proteome analysis by 2-DE and MS revealed 98 significantly deregulated proteins, 44 in GS(+) and 54 in GS(-) tumors. Twelve of these proteins showed expression changes in both tumor types, but only seven of them were deregulated in the same direction. Several of the identified enzymes could be assigned to fundamental metabolic or other cellular pathways with characteristically different alterations in GS(+) and GS(-) tumors such as ammonia and amino acid turnover, cellular energy supply, and calcium homeostasis. Our data suggest that GS(+) and GS(-) tumor cells show a completely different biology and use divergent evolutionary strategies to gain a selective advantage over normal hepatocytes.     

Methylation of histone H3 lysine-79 by Dot1p plays multiple roles in the response to UV damage in Saccharomyces cerevisiae

Various proteins have been found to play roles in both the repair of UV damaged DNA and heterochromatin-mediated silencing in the yeast Saccharomyces cerevisiae. In particular, factors that are involved in the methylation of lysine-79 of histone H3 by Dot1p have been implicated in both processes, suggesting a bipartite function for this modification. We find that a dot1 null mutation and a histone H3 point mutation at lysine-79 cause increased sensitivity to UV radiation, suggesting that lysine-79 methylation is important for efficient repair of UV damage. Epistasis analysis between dot1 and various UV repair genes indicates that lysine-79 methylation plays overlapping roles within the nucleotide excision, post-replication and recombination repair pathways, as well as RAD9-mediated checkpoint function. In contrast, epistasis analysis with the H3 lysine-79 point mutation indicates that the lysine-to-glutamic acid substitution exerts specific effects within the nucleotide excision repair and post-replication repair pathways, suggesting that this allele only disrupts a subset of the functions of lysine-79 methylation. The overall results indicate the existence of distinct and separable roles of histone H3 lysine-79 methylation in the response to UV damage, potentially serving to coordinate the various repair processes.     

Sperm divergence in a passerine contact zone: Indication of reinforcement at the gametic level

Postcopulatory sexual selection may promote evolutionary diversification in sperm form, but the contribution of between‐species divergence in sperm morphology to the origin of reproductive isolation and speciation remains little understood. To assess the possible role of sperm diversification in reproductive isolation, we studied sperm morphology in two closely related bird species, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (Luscinia luscinia), that hybridize in a secondary contact zone spanning Central and Eastern Europe. We found: (1) striking divergence between the species in total sperm length, accompanied by a difference in the length of the mitochondrial sperm component; (2) greater divergence between species in sperm morphology in sympatry than in allopatry, with evidence for character displacement in sperm head length detected in L. megarhynchos; (3) interspecific hybrids showing sperm with a length intermediate between the parental species, but no evidence for decreased sperm quality (the proportion of abnormal spermatozoa in ejaculates). Our results demonstrate that divergence in sperm morphology between the two nightingale species does not result in intrinsic postzygotic isolation, but may contribute to postcopulatory prezygotic isolation. This isolation could be strengthened in sympatry by reinforcement.     

The phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors

Serine proteases are involved in many processes in the nervous system and specific inhibitors tightly control their proteolytic activity. Thrombin is thought to play a role in tissue development and homeostasis. To date, protease nexin-1 is the only known endogenous protease inhibitor that specifically interferes with thrombotic activity and is expressed in the brain. In this study, we report the detection of a novel thrombin inhibitory activity in the brain of protease nexin-1(-/-) mice. Purification and subsequent analysis by tandem mass spectrometry identified this protein as the phosphatidylethanolamine-binding protein (PEBP). We demonstrate that PEBP exerts inhibitory activity against several serine proteases including thrombin, neuropsin, and chymotrypsin, whereas trypsin, tissue type plasminogen activator, and elastase are not affected. Since PEBP does not share significant homology with other serine protease inhibitors, our results define it as the prototype of a novel class of serine protease inhibitors. PEBP immunoreactivity is found on the surface of Rat-1 fibroblast cells and although its sequence contains no secretion signal, PEBP-H(6) can be purified from the conditioned medium upon recombinant expression.     

Effect of rooperol on collagen synthesis and cell growth

The effect of rooperol on type I collagen synthesis in normal skin and lung fibroblasts and cell growth in normal and transformed fibroblasts was investigated. Low concentrations of rooperol selectively inhibited the growth of transformed cells while stimulating collagen synthesis in normal fibroblasts. Elevated collagen synthesis and deposition could impede tumour cell invasion and metastasis, implying that rooperol may be useful as an antimetastatic agent in the treatment of cancer.     

Metabolic engineering of the terpenoid biosynthetic pathway of Escherichia coli for production of the carotenoids β-carotene and zeaxanthin

Metabolic engineering of the early non-mevalonate terpenoid pathway of Escherichia coli was carried out to increase the supply of prenyl pyrophosphates as precursor for carotenoid production. Transformation with the genes dxs for over-expression of 1-deoxy-d-xylulose 5-phosphate synthase, dxr for 1-deoxy-d-xylulose 5-phosphate reductoisomerase and idi encoding an isopentenyl pyrophosphate stimulated carotenogenesis up to 3.5-fold. Co-transformation of idi with either dxs or dxr had an additive effect on ß-carotene and zeaxanthin production which reached 1.6 mg g^–1 dry wt.