12 research outputs found
Periplasmic Cleavage and Modification of the 1-Phosphate Group of Helicobacter Pylori Lipid A
Pathogenic bacteria modify the lipid A portion of their lipopolysaccharide to help evade the host innate immune response. Modification of the negatively charged phosphate groups of lipid A aids in resistance to cationic antimicrobial peptides targeting the bacterial cell surface. The lipid A of Helicobacter pylori contains a phosphoethanolamine (pEtN) unit directly linked to the 1-position of the disaccharide backbone. This is in contrast to the pEtN units found in other pathogenic Gram-negative bacteria, which are attached to the lipid A phosphate group to form a pyrophosphate linkage. This study describes two enzymes involved in the periplasmic modification of the 1-phosphate group of H. pylori lipid A. By using an in vitro assay system, we demonstrate the presence of lipid A 1-phosphatase activity in membranes of H. pylori. In an attempt to identify genes encoding possible lipid A phosphatases, we cloned four putative orthologs of Escherichia coli pgpB, the phosphatidylglycerol-phosphate phosphatase, from H. pylori 26695. One of these orthologs, Hp0021, is the structural gene for the lipid A 1-phosphatase and is required for removal of the 1-phosphate group from mature lipid A in an in vitro assay system. Heterologous expression of Hp0021 in E. coli resulted in the highly selective removal of the 1-phosphate group from E. coli lipid A, as demonstrated by mass spectrometry. We also identified the structural gene for the H. pylori lipid A pEtN transferase (Hp0022). Mass spectrometric analysis of the lipid A isolated from E. coli expressing Hp0021 and Hp0022 shows the addition of a single pEtN group at the 1-position, confirming that Hp0022 is responsible for the addition of a pEtN unit at the 1-position in H. pylori lipid A. In summary, we demonstrate that modification of the 1-phosphate group of H. pylori lipid A requires two enzymatic steps
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Interaction of group IA phospholipase A2 with metal ions and phospholipid vesicles probed with deuterium exchange mass spectrometry.
Deuterium exchange mass spectrometric evaluation of the cobra venom (Naja naja naja) group IA phospholipase A 2 (GIA PLA 2) was carried out in the presence of metal ions Ca (2+) and Ba (2+) and phospholipid vesicles. Novel conditions for digesting highly disulfide bonded proteins and a methodology for studying protein-lipid interactions using deuterium exchange have been developed. The enzyme exhibits unexpectedly slow rates of exchange in the two large alpha-helices of residues 43-53 and 89-101, which suggests that these alpha-helices are highly rigidified by the four disulfide bonds in this region. The binding of Ca (2+) or Ba (2+) ions decreased the deuterium exchange rates for five regions of the protein (residues 24-27, 29-40, 43-53, 103-110, and 111-114). The magnitude of the changes was the same for both ions with the exception of regions of residues 24-27 and 103-110 which showed greater changes for Ca (2+). The crystal structure of the N. naja naja GIA PLA 2 contains a single Ca (2+) bound in the catalytic site, but the crystal structures of related PLA 2s contain a second Ca (2+) binding site. The deuterium exchange studies reported here clearly show that in solution the GIA PLA 2 does in fact bind two Ca (2+) ions. With dimyristoylphosphatidylcholine (DMPC) phospholipid vesicles with 100 microM Ca (2+) present at 0 degrees C, significant areas on the i-face of the enzyme showed decreases in the rate of exchange. These areas included regions of residues 3-8, 18-21, and 56-64 which include Tyr-3, Trp-61, Tyr-63, and Phe-64 proposed to penetrate the membrane surface. These regions also contained Phe-5 and Trp-19, proposed to bind the fatty acyl tails of substrate
Interaction of group IA phospholipase A2 with metal ions and phospholipid vesicles probed with deuterium exchange mass spectrometry.
Deuterium exchange mass spectrometric evaluation of the cobra venom (Naja naja naja) group IA phospholipase A 2 (GIA PLA 2) was carried out in the presence of metal ions Ca (2+) and Ba (2+) and phospholipid vesicles. Novel conditions for digesting highly disulfide bonded proteins and a methodology for studying protein-lipid interactions using deuterium exchange have been developed. The enzyme exhibits unexpectedly slow rates of exchange in the two large alpha-helices of residues 43-53 and 89-101, which suggests that these alpha-helices are highly rigidified by the four disulfide bonds in this region. The binding of Ca (2+) or Ba (2+) ions decreased the deuterium exchange rates for five regions of the protein (residues 24-27, 29-40, 43-53, 103-110, and 111-114). The magnitude of the changes was the same for both ions with the exception of regions of residues 24-27 and 103-110 which showed greater changes for Ca (2+). The crystal structure of the N. naja naja GIA PLA 2 contains a single Ca (2+) bound in the catalytic site, but the crystal structures of related PLA 2s contain a second Ca (2+) binding site. The deuterium exchange studies reported here clearly show that in solution the GIA PLA 2 does in fact bind two Ca (2+) ions. With dimyristoylphosphatidylcholine (DMPC) phospholipid vesicles with 100 microM Ca (2+) present at 0 degrees C, significant areas on the i-face of the enzyme showed decreases in the rate of exchange. These areas included regions of residues 3-8, 18-21, and 56-64 which include Tyr-3, Trp-61, Tyr-63, and Phe-64 proposed to penetrate the membrane surface. These regions also contained Phe-5 and Trp-19, proposed to bind the fatty acyl tails of substrate
Enzymatic Synthesis of Lipid A Molecules with Four Amide-linked Acyl Chains
Richardson House Parsonage (a saltbox house built in 1748 to accommodate the village parson or clergyman), located on the south side of the Common; Old Sturbridge Village (OSV) is a living museum located in Sturbridge, Massachusetts, in the United States, which re-creates life in rural New England during the 1830s. It is the largest living museum in New England, covering more than 200 acres (80 hectares). Located 1 1/2 mile W of town of Sturbridge; town is full-scale reproduction of a 19th-century New England village, complete with general stores, horse-driven vehicles and a meeting house. Source: Wikipedia; http://en.wikipedia.org/wiki/Main_Page (accessed 2/10/2008
Preclinical Characterization of the FAAH Inhibitor JNJ-42165279
The pre-clinical characterization
of the aryl piperazinyl urea
inhibitor of fatty acid amide hydrolase (FAAH) <b>JNJ-42165279</b> is described. <b>JNJ-42165279</b> covalently inactivates the
FAAH enzyme, but is highly selective with regard to other enzymes,
ion channels, transporters, and receptors. <b>JNJ-42165279</b> exhibited excellent ADME and pharmacodynamic properties as evidenced
by its ability to block FAAH in the brain and periphery of rats and
thereby cause an elevation of the concentrations of anandamide (AEA),
oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The compound
was also efficacious in the spinal nerve ligation (SNL) model of neuropathic
pain. The combination of good physical, ADME, and PD properties of <b>JNJ-42165279</b> supported it entering the clinical portfolio
Aryl Piperazinyl Ureas as Inhibitors of Fatty Acid Amide Hydrolase (FAAH) in Rat, Dog, and Primate
A series of aryl piperazinyl ureas that act as covalent
inhibitors
of fatty acid amide hydrolase (FAAH) is described. A potent and selective
(does not inhibit FAAH-2) member of this class, JNJ-40355003, was
found to elevate the plasma levels of three fatty acid amides: anandamide,
oleoyl ethanolamide, and palmitoyl ethanolamide, in the rat, dog,
and cynomolgous monkey. The elevation of the levels of these lipids
in the plasma of monkeys suggests that FAAH-2 may not play a significant
role in regulating plasma levels of fatty acid ethanolamides in primates