Distinct intracellular signaling in tumor necrosis factor-related apoptosis-inducing ligand- and CD95 ligand-mediated apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in tumor cells but not in healthy cells. Similar to CD95 ligand (CD95L), TRAIL signaling requires ligand-receptor interaction; the downstream signaling molecules, such as Fas-associated death domain and caspase-8, also seem similar. Using cells stably expressing TRAIL and CD95L, we show that both TRAIL and CD95L induce apoptosis in the rat colon carcinoma cell line CC531. The mitochondrial damage (loss of mitochondrial membrane potential (MMP) and release of cytochrome c) observed after co-incubation with TRAIL-expressing cells occurs much earlier than that observed with CD95L-expressing cells. The decrease in MMP induced by both ligands was caspase-8-mediated; no difference in caspase-8 activation by TRAIL and CD95L was found. TRAIL, but not CD95L, induced activation of caspase-10. bcl-2 overexpression could not prevent TRAIL-induced mitochondrial dysfunction, whereas it completely prevented CD95L-mediated loss of MMP and cytochrome c release. The selective effect of TRAIL on tumor cells and the apparent inability of bcl-2 to block TRAIL-induced apoptosis suggest that TRAIL may offer a lead for cancer therapy in the future.     

Immunohistochemical demonstration of -(carboxymethyl)lysine protein adducts in normal and osteoarthritic cartilage

N(epsilon)-(carboxymethyl)lysine (CML) is an advanced glycation end product formed by non-enzymatic glycation and oxidation of proteins. The distribution pattern of CML-modified proteins in normal and osteoarthritic (OA) cartilage was investigated using specific antibodies. In healthy articular cartilage, immunoreactivity for CML was preferably found in the extracellular matrix (ECM) of the superficial layer. In OA samples, CML immunoreactivity was not restricted to the ECM of the superficial layer. Interestingly, OA chondrocytes showed a remarkable cytoplasmic immunoreactivity for CML. With the help of a western blot analysis CML-modified proteins between 68 and 39 kDa could be demonstrated in OA cartilage samples. These results suggest that the accumulation of CML adducts contributes to the matrix damage in osteoarthritis. Therefore, the inhibition of CML accumulation may represent an effective therapeutic strategy to prevent severe OA cartilage injury.     

Multi parameter in vitro testing of ratjadone using flow cytometry

Ratjadone, isolated from the myxobacterium Sorangium cellulosum, belongs to the family of so-called orphan ligands, which includes leptomycin, callystatin and other compounds. In previous screening tests, ratjadone revealed a growth inhibitory effect against bacteria, yeast and human cancer cells. Following these first results, ratjadone was tested on several human tumour cell lines (Jurkat, HepG2, U87-MG) and, as a control, on a non-tumour cell line (RLC18) for its mode of action. The cell analysis was carried out by flow cytometry. This comprised cell density measurements, live-dead analysis, cell-cycle analysis and detection of apoptosis. First experiments confirmed the growth inhibitory effect on any chosen tumour cell line. Following these results a dose effect relationship was monitored, confirming the high effectiveness of ratjadone against cell growth at nanomolar concentration. Cell cycle analysis has shown that ratjadone intervenes in the cell cycle by arresting the cells in G1-phase. Biological testing of additional ratjadone derivatives with changed configuration and stereochemistry, identified the pharmacophoric site of the molecule.     

GluR2 ligand-binding core complexes: importance of the isoxazolol moiety and 5-substituent for the binding mode of AMPA-type agonists

X-ray structures of the GluR2 ligand-binding core in complex with (S)-Des-Me-AMPA and in the presence and absence of zinc ions have been determined. (S)-Des-Me-AMPA, which is devoid of a substituent in the 5-position of the isoxazolol ring, only has limited interactions with the partly hydrophobic pocket of the ligand-binding site, and adopts an AMPA-like binding mode. The structures, in comparison with other agonist complex structures, disclose the relative importance of the isoxazolol ring and of the substituent in the 5-position for the mode of binding. A relationship appears to exist between the extent of interaction of the ligand with the hydrophobic pocket and the affinity of the ligand.     

A unique structural pattern shared by T-cell-activating and abscess-regulating zwitterionic polysaccharides

In contrast to the conventional dogma that carbohydrates are poorly immunogenic T-cell-independent antigens, zwitterionic polysaccharides (ZPSs) can significantly stimulate T-cell proliferation and regulate abscess formation in bacterial infection. Despite their similar biological activities, ZPSs from various bacteria are greatly different in primary chemical compositions and building block linkages. To identify the common structural features that govern the peculiar immunologic activity of ZPSs, we have been determining three-dimensional structures of compositionally different ZPSs by NMR spectroscopy and molecular mechanics and dynamics calculations. We report here the conformation of type 1 capsular polysaccharide from the human pathogen Streptococcus pneumoniae (Sp1) to be a right-handed helix with repeated zwitterionically charged grooves. We also report the striking similarity between the structures of Sp1 and our previously determined PS A2 from Bacteroides fragilis. These results support our hypothesis that T-cell-activating ZPSs assume similar conformational and charge patterns that are recognized by specific receptors and that account for their common property as T-cell activators.     

Role of the N-terminal extension of the (betaalpha)8-barrel enzyme indole-3-glycerol phosphate synthase for its fold, stability, and catalytic activity

Indole-3-glycerol phosphate synthase (IGPS) catalyzes the fifth step in the biosynthesis of tryptophan. It belongs to the large and versatile family of (betaalpha)(8)-barrel enzymes but has an unusual N-terminal extension of about 40 residues. Limited proteolysis with trypsin of IGPS from both Sulfolobus solfataricus (sIGPS) and Thermotoga maritima (tIGPS) removes about 25 N-terminal residues and one of the two extra helices contained therein. To assess the role of the extension, the N-terminally truncated variants sIGPSDelta(1-26) and tIGPSDelta(1-25) were produced recombinantly in Escherichia coli, purified, and characterized in comparison to the wild-type enzymes. Both sIGPSDelta(1-26) and tIGPSDelta(1-25) have unchanged oligomerization states and turnover numbers. In contrast, their Michaelis constants for the substrate 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate are increased, and their resistance toward unfolding induced by heat and guanidinium chloride is decreased. sIGPSDelta(1-26) was crystallized, and its X-ray structure was solved at 2.8 A resolution. The comparison with the known structure of sIGPS reveals small differences that account for its reduced substrate affinity and protein stability. The structure of the core of sIGPSDelta(1-26) is, however, unchanged compared to sIGPS, explaining its retained catalytic activity and consistent with the idea that it evolved from the same ancestor as the phosphoribosyl anthranilate isomerase and the alpha-subunit of tryptophan synthase. These (betaalpha)(8)-barrel enzymes catalyze the reactions preceding and following IGPS in tryptophan biosynthesis but lack an N-terminal extension.     

The Lin12-notch repeats of pregnancy-associated plasma protein-A bind calcium and determine its proteolytic specificity

The Lin12-Notch repeat (LNR) module of about 35 residues is a hallmark of the Notch receptor family. Three copies, arranged in tandem, are invariably present in the extracellular portion of the Notch receptors. Although their function is unknown, genetic and biochemical data indicate that the LNR modules participate in the regulation of ligand-induced proteolytic cleavage of the Notch receptor, a prerequisite to intramembrane cleavage and Notch signaling. Outside the Notch receptor family, the LNR module is present only in the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) and its homologue PAPP-A2, which also contain three copies. Curiously, LNR modules 1 and 2 are present within the proteolytic domain of PAPP-A/A2, but LNR3 is separated from LNR2 by more than 1000 amino acids. The growth factor antagonists insulin-like growth factor-binding protein (IGFBP)-4 and -5 are both substrates of PAPP-A. We provide here evidence that the PAPP-A LNR modules function together to determine the proteolytic specificity of PAPP-A. Analysis of C-terminally truncated PAPP-A mutants followed by the analysis of LNR deletion mutants demonstrated that each of the three PAPP-A LNR modules is strictly required for proteolytic activity against IGFBP-4 but not for proteolytic activity against IGFBP-5. Individual substitution of conserved LNR residues predicted to participate in calcium coordination caused elimination (D341A, D356A, D389A, D1484A, D1499A, and D1502A) or a significant reduction (D359A and E392A) of IGFBP-4 proteolysis, whereas IGFBP-5 proteolysis was unaffected. The activity of the latter mutants against IGFBP-4 could be partially rescued by calcium, and the addition of the calcium-binding protein calbindin D9k to wild-type PAPP-A eliminated activity against IGFBP-4 but not against IGFBP-5, demonstrating that the PAPP-A LNR modules bind calcium ions. We propose a model in which LNR3 is spatially localized in proximity to LNR1 and -2, forming a single functional unit.     

Visualization of large-scale correlations in gene expressions

Large-scale expression data are today measured for several thousands of genes simultaneously. Furthermore, most genes are being categorized according to their properties. This development has been followed by an exploration of theoretical tools to integrate these diverse data types. A key problem is the large noise-level in the data. Here, we investigate ways to extract the remaining signals within these noisy data sets. We find large-scale correlations within data from Saccharomyces cerevisiae with respect to properties of the encoded proteins. These correlations are visualized in a way that is robust to the underlying noise in the measurement of the individual gene expressions. In particular, for S. cerevisiae we observe that the proteins corresponding to the 400 highest expressed genes typically are localized to the cytoplasm. These most expressed genes are not essential for cell survival.