Structural insights into molecular function of the metastasis-associated phosphatase PRL-3.

Phosphatases and kinases are the cellular signal transduction enzymes that control protein phosphorylation. PRL phosphatases constitute a novel class of small (20 kDa), prenylated phosphatases with oncogenic activity. In particular, PRL-3 is consistently overexpressed in liver metastasis in colorectal cancer cells and represents a new therapeutic target. Here, we present the solution structure of PRL-3, the first structure of a PRL phosphatase. The structure places PRL phosphatases in the class of dual specificity phosphatases with closest structural homology to the VHR phosphatase. The structure, coupled with kinetic studies of site-directed mutants, identifies functionally important residues and reveals unique features, differentiating PRLs from other phosphatases. These differences include an unusually hydrophobic active site without the catalytically important serine/threonine found in most other phosphatases. The position of the general acid loop indicates the presence of conformational change upon catalysis. The studies also identify a potential regulatory role of Cys(49) that forms an intramolecular disulfide bond with the catalytic Cys(104) even under mildly reducing conditions. Molecular modeling of the highly homologous PRL-1 and PRL-2 phosphatases revealed unique surface elements that are potentially important for specificity

Novel, acid-labile, hydroxydiether lipid cores in methanogenic bacteria.

Polar ether lipids extracted from 15 methanogenic bacteria, representative of seven genera, were screened by nuclear magnetic resonance and thin layer chromatography for the presence of hydroxyl groups on the C20-phytanyl moieties. Major amounts of hydroxydiether core lipid were confirmed for Methanosaeta concilii and discovered in two Methanosarcina species, Methanococcus voltae, and tentatively in several Methanobacterium species. Signals at 1.24 and 1.8-1.9 ppm in 1H NMR spectra are characteristic of Methanosaeta concilii lipids hydroxylated on carbon-3 (sn-3 chain). Related signals, which were shifted slightly, appeared in spectra of the polar lipids extracted from both Methanosarcina species. Following mild hydrolysis to remove the polar head groups, only two chromatographically distinct core lipids were found in significant amounts in Methanosarcina barkeri (and Methanosarcina mazei) consisting of 43% 2,3-di-O-phytanyl-sn-glycerol (C20,20-diether) and 57% C20,20-hydroxydiether. This latter core lipid differed from the hydroxydiether from M. concilii by hydroxylation, on carbon-3, of the phytanyl chain in ether linkage to the sn-2 carbon of glycerol. The structural assignment was based on identification of the novel hydroxydiether core and its methylation products by 1H NMR, 13C NMR, and mass spectroscopy. The hydroxy core lipid degraded to various products during standard methanolic HCl and sulfuric acid procedures, including a methoxy derivative (methanolic HCl) and the 3-mono-O-phytanyl-sn-glycerol

Structures of minor ether lipids isolated from the aceticlastic methanogen, Methanothrix concilii GP6.

Structures were determined for two phospholipids and three glycolipids purified from chloroform-methanol extracts of Methanothrix concilii GP6. Together they accounted for 14% of the total lipid and were based on a C20,20-diether core structure consisting of either 2,3-di-O-phytanyl-sn-glycerol or its 3'-hydroxy analog, namely, 2-O-[3,7,11,15-tetramethylhexadecyl]-3-O-[3'- hydroxy-3',7',11',15'-tetramethylhexadecyl]-sn-glycerol. These two core lipids formed phosphodiester bonds to ethanolamine and glycosidic bonds to beta-D-galactopyranose. A third glycolipid consisted of the triglycosyl head group beta-D-galactopyranosyl-(1----6)-[beta-D-glucopyranosyl-(1----3)]-beta-D - galactopyranose in glycosidic linkage to the 3'-hydroxydiether core lipid

Folding-related dimerization of human cystatin C

NRC publication: Ye

Folding related dimerization of human cystatin C

With the aim to improve our understanding of the structural basis for protein self-association and aggregation, in particular in relationship to protein refolding and amyloid formation, folding-related processes for human cystatin C have been studied. Using NMR spectroscopy together with chromatographic and electrophoretic methods, a self-association process resulting in dimer formation for protein samples treated with denaturing agents as well as for samples subjected to low pH or high temperature conditions could be studied with amino acid resolution. In all three cases, the dimerization involves properly folded molecules and proceeds via the reactive site of the inhibitor, which leads to complete loss of its biological activity. This dimerization process has potential relevance for amyloid formation by the brain hemorrhage-causing Leu-Gln variant of cystatin C. The results also indicate that cystatin C dimerization and inactivation may occur in acidified compartments in vivo, which could be relevant for the physiological regulation of cysteine proteinase activity

Identification of degradation artifacts formed upon treatment of hydroxydiether lipids from methanogens with methanolic-HCl: Can.J.Microbiol.

NRC publication: Ye

H(C)CH-COSY and (H)CCH-COSY experiments for 13C-labeled proteins in H2O solution

UI - 99018160NRC publication: Ye

Solution structure of the PDZ2 domain from human phosphatase hPTP1E and its interactions with C-terminal peptides from the Fas receptor

NRC publication: Ye

Solution structure of the PDZ2 domain from human phosphatase hPTP1E and its interactions with C-terminal peptides from the Fas receptor

NRC publication: Ye