Structural basis and kinetics of DsbD-dependent cytochrome c maturation

DsbD from Escherichia coli transports two electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of an N-terminal periplasmic domain (nDsbD), a C-terminal periplasmic domain, and a central transmembrane domain. Each domain possesses two cysteines required for electron transport. Herein, we demonstrate fast (3.9 x 10(5) M(-1)s(-1)) and direct disulfide exchange between nDsbD and CcmG, a highly specific disulfide reductase essential for cytochrome c maturation. We determined the crystal structure of the disulfide-linked complex between nDsbD and the soluble part of CcmG at 1.94 A resolution. In contrast to the other two known complexes of nDsbD with target proteins, the N-terminal segment of nDsbD contributes to specific recognition of CcmG. This and other features, like the possibility of using an additional interaction surface, constitute the structural basis for the adaptability of nDsbD to different protein substrates.     

Inefficient maturation of the rat luteinizing hormone receptor. A putative way to regulate receptor numbers at the cell surface

Increasing evidence suggests that the folding and maturation of monomeric proteins and assembly of multimeric protein complexes in the endoplasmic reticulum (ER) may be inefficient not only for mutants that carry changes in the primary structure but also for wild type proteins. In the present study, we demonstrate that the rat luteinizing hormone receptor, a G protein-coupled receptor, is one of these proteins that matures inefficiently and appears to be very prone to premature degradation. A substantial portion of the receptors in stably transfected human embryonic kidney 293 cells existed in immature form of M(r) 73,000, containing high mannose-type N-linked glycans. In metabolic pulse-chase studies, only approximately 20% of these receptor precursors were found to gain hormone binding ability and matured to a form of M(r) 90,000, containing bi- and multiantennary sialylated N-linked glycans. The rest had a propensity to form disulfide-bonded complexes with a M(r) 120,000 protein in the ER membrane and were eventually targeted for degradation in proteasomes. The number of membrane-bound receptor precursors increased when proteasomal degradation was inhibited, and no cytosolic receptor forms were detected, suggesting that retrotranslocation of the misfolded/incompletely folded receptors is tightly coupled to proteasomal function. Furthermore, a proteasomal blockade was found to increase the number of receptors that were capable of hormone binding. Thus, these results raise the interesting possibility that luteinizing hormone receptor expression at the cell surface may be controlled at the ER level by regulating the number of newly synthesized proteins that will mature and escape the ER quality control and premature degradation.     

Identification of Tyr900 in the kinase domain of c-Kit as a Src-dependent phosphorylation site mediating interaction with c-Crk

We have previously demonstrated that ligand-stimulation of c-Kit induces phosphorylation of Tyr568 and Tyr570 in the juxtamembrane region of the receptor, leading to recruitment, phosphorylation and activation of members of the Src family of tyrosine kinases. In this paper, we demonstrate that members of the Src family of tyrosine kinases are able to phosphorylate c-Kit selectively on one particular tyrosine residue, Tyr900, located in the second part of the tyrosine kinase domain. In order to identify potential docking partners of Tyr900, a synthetic phosphopeptide corresponding to the amino acid sequence surrounding Tyr900 was used as an affinity matrix. By use of MALDI-TOF mass spectrometry, CrkII was identified as a protein that specifically bound to Tyr900 in a phosphorylation dependent manner, possibly via the p85 subunit of PI3-kinase. Expression of a mutant receptor where Tyr900 had been replaced with a phenylalanine residue (Y900F) resulted in a receptor with reduced ability to phosphorylate CrkII. Together these data support a model where c-Src phosphorylates the receptor, thereby creating docking sites for SH2 domain containing proteins, leading to recruitment of Crk to the receptor.     

Use of 16S ribosomal DNA for delineation of marine bacterioplankton species

All of the marine bacterioplankton-derived 16S ribosomal DNA sequences previously deposited in GenBank were reanalyzed to determine the number of bacterial species in the oceanic surface waters. These sequences have been entered into the database since 1990. The rate of new additions reached a peak in 1999 and subsequently leveled off, suggesting that much of the marine microbial species richness has been sampled. When the GenBank sequences were dereplicated by using 97% similarity as a cutoff, 1,117 unique ribotypes were found. Of the unique sequences, 609 came from uncultured environmental clones and 508 came from cultured bacteria. We conclude that the apparent bacterioplankton species richness is relatively low.     

The evolutionary origins of three new Neotyphodium endophyte species from grasses indigenous to the Southern Hemisphere

Members of the genus Neotyphodium are asexual, seedborne, protective fungal endophytes of cool season grasses that have likely evolved either directly from sexual Epichlo?; species, or by the interspecific hybridization of distinct lineages of Epichlo?; and Neotyphodium. We investigated the evolutionary origins of Neotyphodium endophytes from several grasses that are indigenous to the Southern Hemisphere using a multiple-gene phylogenetic approach. Intron regions of the genes encoding β-tubulin (tub2), translation elongation factor 1-α (tef1) and actin (act1) were amplified by polymerase chain reaction and sequenced. Phylogenetic analyses of these sequences, aligned with homologous sequences from Epichlo?; spp., revealed the evolutionary origins of the Southern Hemisphere endophytes, where one lineage of apparently non-hybrid origin, and three lineages of unique interspecific hybrid origin were identified. On the basis of morphology, host range and evolutionary history, we propose three new species of Neotyphodium. Neotyphodium aotearoae was isolated from Echinopogon ovatus populations from New Zealand and Australia, and comprised a unique, apparently non-hybrid lineage within the Epichlo?; species phylogeny. In contrast, an interspecific hybrid lineage was identified from two Australian Ec. ovatus populations, whose ancestry apparently involved lineages closely related to extant E. festucae and an E. typhina genotype similar to that of isolates from Poa pratensis. Endophytes infecting South African Melica racemosa and M. decumbens (dronkgras) appeared to be hybrids of E. festucae and N. aotearoae or close relatives. The names N. australiense and N. melicicola are proposed for these two hybrid lineages, respectively. The origin of N. tembladerae, an established endophyte species from South American Poa and Festuca spp., was also investigated. Neotyphodium tembladerae appeared to be of hybrid origin, involving E. festucae and an E. typhina genotype similar to that of isolates from Poa nemoralis. The results of this study highlight the widespread occurrence of interspecific hybrid Neotyphodium lineages on a global scale, and the extent of endophyte gene-flow between the Northern and Southern Hemispheres.     

Stereospecific pH-dependent degradation kinetics of R- and S-naproxen-beta-l-O-acyl-glucuronide

The hydrolysis and acyl migration of biosynthetic S-naproxen-beta-l-O-acyl glucuronide (I) and R-naproxen-beta-l-O-acyl glucuronide (II) was followed by HPLC. Nine first-order kinetic rate constants for the hydrolysis and acyl migration between the beta-l-O-acyl glucuronide, its alpha/beta-2, alpha/beta-3-, alpha/beta-4-, and alpha-1-O-acyl isomers and naproxen aglycone were determined for I and II at pH 7.00, 7.40 and 8.00 at 37 degrees C by kinetic simulation. For I the 3-O-acyl isomer was the most stable isomer as the pseudo-equilibrium ratio for the major acyl-migrated isomers was 1:1.5:0.9 (2-O-acyl isomer:3-O-acyl isomer:4-O-acyl isomer). The 3- and 4-O-acyl isomers of II were equally stable as the pseudo-equilibrium ratio for the major acyl-migrated isomers was 1:1.4:1.4 (2-O-acyl isomer:3-O-acyl isomer:4-O-acyl isomer). For both I and II, the pseudo-equilibrium ratio between the major 2-O-acyl isomer and the minor alpha-l-O-acyl isomer was 10:1 (2-O-acyl isomer:alpha-l-O-acyl isomer). The pseudo-equilibrium found for the major acyl-migrated isomers of I and II in the present study corresponds with the pattern previously published for R- and S-ketoprofen-beta-l-O-acyl glucuronide acyl-migrated isomers, suggesting that these findings may be general for acyl-migrated beta-l-O-acyl glucuronides of enantiomeric 2-arylpropionic acids.     

Osteoglycin expression and localization in rabbit tissues and atherosclerotic plaques

The localization of osteoglycin (OG), one of the corneal keratan sulfate proteoglycans, was studied in different normal rabbit tissues, as well as in atherosclerotic lesions, by means of in situ hybridization and immunohistochemistry. OG was associated with the vasculature of all the organs analyzed. Normal aortas showed abundance of the protein in the adventitia and focally in the media. Peripheral vessels showed OG localized only in the adventitia. OG mRNA was restricted to vascular smooth muscle cells, pericytes, and fibroblasts in aorta and skeletal muscle. In striated muscle, OG was abundant and distributed in foci around muscles and vessels, whereas in visceral muscle, the protein was homogeneously distributed throughout the extracellular matrix. In all the other organs studied, OG was only associated with the vasculature, with the exception of the lung and liver. In these two organs, the protein accumulated also around cartilage, alveoli, and hepatic duct. In atherosclerotic lesions, OG mRNA was down-regulated in the media and up-regulated in the activated endothelium and thick neo-intima, whereas the protein accumulated in the front edge of migrating smooth muscle cells. We conclude that OG is a basic component of the vascular extracellular matrix. OG also plays a role in atherosclerosis, and might be useful for therapeutic interventions. In addition, the possible involvement of OG in maintaining physical properties of tissues is discussed.