COPS--cis/trans peptide bond conformation prediction of amino acids on the basis of secondary structure information

SUMMARY: COPS predicts for all 20 naturally occurring amino acids whether the peptide bond in a protein is in cis or trans conformation. The algorithm is based only on secondary structure information of amino acid triplets without considering the amino acid sequence information. Conformation parameters are derived from solved 3D structures deposited in the PDB and led to propensities based on modified Chou-Fasman parameters. COPS analyses amino acid triplets taking only their respective secondary structure into consideration and upon application of a set of rules utilizing the conformation parameters, the N-terminal peptide bond conformation of the middle residue is predicted. COPS was tested on a random selection of protein datasets. AVAILABILITY: The COPS program and further information are freely available from the FMP website at http://www.fmp-berlin.de/nmr/cops CONTACT: labudde@fmp-berlin.de.     

Adrenodoxin (Adx) and CYP11A1 (P450scc) induce apoptosis by the generation of reactive oxygen species in mitochondria

Mitochondrial cytochrome P450 systems are an indispensable component of mammalian steroid biosynthesis; they catalyze regio- and stereo-specific steroid hydroxylations and consist of three protein entities: adrenodoxin reductase (AdR), adrenodoxin (Adx), and a mitochondrial cytochrome P450 enzyme, e.g., CYP11A1 (P450 side chain cleavage, P450scc). It is known that the latter two are able to generate reactive oxygen species (ROS) in vitro . In this study, we investigated whether this ROS generation also occurs in vivo and, if so, whether it leads to the induction of apoptosis. We found that overexpression of either human or bovine Adx causes a significant loss of viability in 11 different cell lines. This loss of viability does not depend on the presence of the tumor suppressor protein p53. Transient overexpression of human Adx in HCT116 cells leads to ROS production, to a disruption of the mitochondrial transmembrane potential (DeltaPsi), to cytochrome c release from the mitochondria, and to caspase activation. In contrast, the effect of transient overexpression of human CYP11A1 on cell viability varies in different cell lines, with some being sensitive and others not. We conclude that mitochondrial cytochrome P450 systems are a source of mitochondrial ROS production and can play a role in the induction of mitochondrial apoptosis.     

Functional importance of the C-terminus of the human norepinephrine transporter

Three C-terminal variants of the human norepinephrine transporter (hNET) are known: the wild-type hNET in which exon 14 encodes the last seven amino acids and two variants with either three or 18 amino acids encoded by an alternatively spliced exon 15. In transfected HEK293 cells we compared by means of [(3)H]norepinephrine ([(3)H]NE) uptake and [(3)H]nisoxetine ([(3)H]NIS) binding the functional properties of the wild-type hNET with those of the more abundant long splice variant containing exon 15 (hNET-Ex15L) and of two artificial hNET mutants lacking either the last three (hNET-Ex14-4) or all seven (hNET-Ex14-0) C-terminal amino acids of exon 14. No differences among the NET isoforms were observed concerning the K(m) for uptake of NE and the K(D) for binding of NIS. However, compared with the wild-type hNET, the three isoforms (hNET-Ex15L, hNET-Ex14-4 and hNET-Ex14-0) showed a pronounced decrease in V(max) of [(3)H]NE uptake and B(max) of [(3)H]NIS binding which correlated with strongly reduced surface expression of the transporter isoforms. The decrease in surface expression of the hNET isoforms is probably a consequence of the lack of the three amino acids leucine, alanine and isoleucine at the C-terminal end which may represent a motif facilitating cell surface expression of the hNET. Expression of hNET-Ex15L exerted a dominant negative effect on plasma membrane expression of the wild-type hNET and thus may represent a novel mechanism for regulation of noradrenergic neurotransmission.     

Sex Steroids Inhibit Osmotic Swelling of Retinal Glial Cells

Osmotic swelling of glial cells may contribute to the development of retinal edema. We investigated whether sex steroids inhibit the swelling of glial somata in acutely isolated retinal slices and glial cells of the rat. Superfusion of retinal slices or cells from control animals with a hypoosmolar solution did not induce glial swelling, whereas glial swelling was observed in slices of postischemic and diabetic retinas. Progesterone, testosterone, estriol, and 17ß-estradiol prevented glial swelling with half-maximal effects at approximately 0.3, 0.6, 6, and 20 μM, respectively. The effect of progesterone was apparently mediated by transactivation of metabotropic glutamate receptors, P2Y₁, and adenosine A₁ receptors. The data suggest that sex steroids may inhibit cytotoxic edema in the retina.     

Inhibition of voltage-gated K+ channels in dendritic cells by rapamycin

Rapamycin, an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR), is a widely used immunosuppressive drug. Rapamycin affects the function of dendritic cells (DCs), antigen-presenting cells participating in the initiation of primary immune responses and the establishment of immunological memory. Voltage-gated K(+) (Kv) channels are expressed in and impact on the function of DCs. The present study explored whether rapamycin influences Kv channels in DCs. To this end, DCs were isolated from murine bone marrow and ion channel activity was determined by whole cell patch clamp. To more directly analyze an effect of mTOR on Kv channel activity, Kv1.3 and Kv1.5 were expressed in Xenopus oocytes with or without the additional expression of mTOR and voltage-gated currents were determined by dual-electrode voltage clamp. As a result, preincubation with rapamycin (0-50 nM) led to a gradual decline of Kv currents in DCs, reaching statistical significance within 6 h and 50 nM of rapamycin. Rapamycin accelerated Kv channel inactivation. Coexpression of mTOR upregulated Kv1.3 and Kv1.5 currents in Xenopus oocytes. Furthermore, mTOR accelerated Kv1.3 channel activation and slowed down Kv1.3 channel inactivation. In conclusion, mTOR stimulates Kv channels, an effect contributing to the immunomodulating properties of rapamycin in DCs.     

Differentiation-associated expression of ceramidase isoforms in cultured keratinocytes and epidermis

Ceramides (Cers) accumulate within the interstices of the outermost epidermal layers, or stratum corneum (SC), where they represent critical components of the epidermal permeability barrier. Although the SC contains substantial sphingol, indicative of ceramidase (CDase) activity, which CDase isoforms are expressed in epidermis remains unresolved. We hypothesized here that CDase isoforms are expressed within specific epidermal compartments in relation to functions that localize to these layers. Keratinocytes/epidermis express all five known CDase isoforms, of which acidic and alkaline CDase activities increase significantly with differentiation, persisting into the SC. Conversely, neutral and phytoalkaline CDase activities predominate in proliferating keratinocytes. These differentiation-associated changes in isoform activity/protein are attributed to corresponding, differentiation-associated changes in mRNA levels (by quantitative RT-PCR). Although four of the five known CDase isoforms are widely expressed in cutaneous and extracutaneous tissues, alkaline CDase-1 occurs almost exclusively in epidermis. These results demonstrate large, differentiation-associated, and tissue-specific variations in the expression and activities of all five CDase isoforms. Because alkaline CDase-1 and acidic CDase are selectively upregulated in the differentiated epidermal compartment, they could regulate functions that localize to the distal epidermis, such as permeability barrier homeostasis and antimicrobial defense.     

Essential role of Isd11 in mitochondrial iron-sulfur cluster synthesis on Isu scaffold proteins

Mitochondria are indispensable for cell viability; however, major mitochondrial functions including citric acid cycle and oxidative phosphorylation are dispensable. Most known essential mitochondrial proteins are involved in preprotein import and assembly, while the only known essential biosynthetic process performed by mitochondria is the biogenesis of iron-sulfur clusters (ISC). The components of the mitochondrial ISC-assembly machinery are derived from the prokaryotic ISC-assembly machinery. We have identified an essential mitochondrial matrix protein, Isd11 (YER048w-a), that is found in eukaryotes only. Isd11 is required for biogenesis of cellular Fe/S proteins and thus is a novel subunit of the mitochondrial ISC-assembly machinery. It forms a complex with the cysteine desulfurase Nfs1 and is required for formation of an Fe/S cluster on the Isu scaffold proteins. We conclude that Isd11 is an indispensable eukaryotic component of the mitochondrial machinery for biogenesis of Fe/S proteins.     

Microgravity dependence of excitable biological and physicochemical media

Neuronal tissue and especially the central nervous system (CNS) is an excitable medium. Self-organisation, pattern formation, and propagating excitation waves as typical characteristics in excitable media consequently have been found in neuronal tissue. The properties of such phenomena in excitable media do critically depend on the parameters (i.e., electromagnetic fields, temperature, chemical drugs) of the system and on small external forces to which gravity belongs. The spreading depression, a propagating excitation depression wave of neuronal activity, is one of the best described of the those wave phenomena in the CNS. Especially in the retina as a true part of the CNS it can be easily observed with optical techniques due to the high intrinsic optical signal of this tissue. Another of such waves in neuronal tissue is the propagating action potential in nerve fibres. In this paper, data from our laboratories concerning the influence of gravity on the velocity of propagating waves in excitable media are summarized mainly in terms of the retinal spreading depression and propagating action potentials. Additionally, we have used waves in gels of the Belousov-Zhabotinsky reaction as the physicochemical model system of biological activity as the properties of these waves follow the same theories as the spreading depression and action potentials and they have some striking similarities in wave behavior. Thus propagating Belousov-Zhabotinsky waves are described by their gravity dependence.     

Signaling pathways involved in PDGF-evoked cellular responses in human RPE cells

We examined whether PDGF may directly stimulate the expression of VEGF by retinal pigment epithelial (RPE) cells in vitro, and the involvement of three signal transduction pathways in the regulation of PDGF-evoked cell proliferation, migration, and production of VEGF-A was investigated. PDGF stimulated the gene and protein expression of VEGF-A by RPE cells, and increased cell proliferation and chemotaxis. PDGF activated all signaling pathways investigated, as determined by increased phosphorylation levels of ERK1/2, p38, and Akt proteins. The three signaling pathways were involved in the mediation of PDGF-evoked cell proliferation, while p38 and PI3K mediated cell migration, and PI3K mediated secretion of VEGF-A. In addition to VEGF-A, the cells expressed mRNAs for various members of the VEGF family and for their receptors, including VEGF-B, -C, -D, flt-1, and KDR. The data indicate that PDGF selectively stimulates the expression of VEGF-A in RPE cells. PDGF evokes at least three signal transduction pathways which are differentially involved in various cellular responses.     

Emerging roles of DP and CRTH2 in allergic inflammation

The lipid mediator prostaglandin D(2) (PGD(2)) has long been implicated in various inflammatory diseases including asthma. PGD(2) elicits biological responses by activating two seven-transmembrane (7TM) G-protein-coupled receptors, the D-prostanoid receptor DP and the chemoattractant receptor homologous-molecule expressed on T-helper-type-2 cells (CRTH2), which are linked to different signaling pathways. Understanding how immune cells integrate and coordinate signals that are triggered by the same ligand is crucial for the development of novel anti-inflammatory therapies. Here, we examine the roles of DP and CRTH2 in the orchestration of complex inflammatory processes, and discuss their importance as emerging targets for the treatment of asthma and inflammatory diseases.