Nociceptin/orphanin FQ and drugs of abuse

Nociceptin/orphanin FQ (NC) binds with high affinity to the opioid receptor-like1 (ORL1) receptor. NC has been reported to block opioid-induced supraspinal analgesia, and it has been proposed that it may represent a functional antiopioid peptide in the control of brain nociceptive processes. The wide distribution of NC and of its receptors in the central nervous system suggests, however, that it may be involved in the control of a variety of biologic functions. Increasing evidence indicates that it may influence the rewarding and reinforcing properties of drugs of abuse. NC has been shown to abolish the rewarding properties of ethanol and morphine in the place conditioning paradigm, to reduce ethanol consumption in alcohol-preferring rats and to inhibit stress-induced alcohol-seeking behavior. These findings suggest that drugs directed at central NC receptors may represent an interesting approach to the treatment of ethanol and opiate abuse.     

Data reduction of isotope-resolved LC-MS spectra

MOTIVATION: Data reduction of liquid chromatography-mass spectrometry (LC-MS) spectra can be a challenge due to the inherent complexity of biological samples, noise and non-flat baseline. We present a new algorithm, LCMS-2D, for reliable data reduction of LC-MS proteomics data. RESULTS: LCMS-2D can reliably reduce LC-MS spectra with multiple scans to a list of elution peaks, and subsequently to a list of peptide masses. It is capable of noise removal, and deconvoluting peaks that overlap in m/z, in retention time, or both, by using a novel iterative peak-picking step, a 'rescue' step, and a modified variable selection method. LCMS-2D performs well with three sets of annotated LC-MS spectra, yielding results that are better than those from PepList, msInspect and the vendor software BioAnalyst. AVAILABILITY: The software LCMS-2D is available under the GNU general public license from http://www.bioc.aecom.yu.edu/labs/angellab/as a standalone C program running on LINUX.     

BACE1 cytoplasmic domain interacts with the copper chaperone for superoxide dismutase-1 and binds copper

The amyloidogenic pathway leading to the production and deposition of Abeta peptides, major constituents of Alzheimer disease senile plaques, is linked to neuronal metal homeostasis. The amyloid precursor protein binds copper and zinc in its extracellular domain, and the Abeta peptides also bind copper, zinc, and iron. The first step in the generation of Abeta is cleavage of amyloid precursor protein by the aspartic protease BACE1. Here we show that BACE1 interacts with CCS (the copper chaperone for superoxide dismutase-1 (SOD1)) through domain I and the proteins co-immunoprecipitate from rat brain extracts. We have also been able to visualize the co-transport of membranous BACE1 and soluble CCS through axons. BACE1 expression reduces the activity of SOD1 in cells consistent with direct competition for available CCS as overexpression of CCS restores SOD1 activity. Finally, we demonstrate that the twenty-four residue C-terminal domain of BACE1 binds a single Cu(I) atom with high affinity through cysteine residues.     

Surface activity and interaction of StarD7 with phospholipid monolayers

StarD7 protein forms stable Gibbs and Langmuir monolayers at the air-buffer interface showing marked surface activity. The latter is enhanced by penetration into phospholipid films at an initial surface pressure above the protein’s own equilibrium adsorption surface pressure to a lipid-free interface. The protein-phospholipid stabilizing interactions at the interface depend on the lipid, with preference for phosphatidylserine, cholesterol, and phosphatidylglycerol, and the increases of lateral surface pressure generated are comparable to those of other membrane-active proteins. The surface activity of StarD7 is strong enough to thermodynamically drive and retain StarD7 at the lipid membrane interface where it may undergo lipid-dependent reorganization as indicated by changes of surface pressure and electrostatics.     

Relationship Between the Nerve Growth Factor-Regulated Clone 73 Gene Product and the 58-Kilodalton Neuronal Intermediate Filament Protein (Peripherin)

Exposure of PC12 cells to nerve growth factor (NGF) has been shown to induce an mRNA that encodes a novel neuronal intermediate filament protein. The findings presented here concern the identity of this filament protein. The major protein in NGF-treated PC12 cell cytoskeletons derived by extraction with 1% Triton X-100 is of apparent Mr = 58,000, focuses by isoelectric focusing as several closely spaced spots of pl 5.6-5.8, and is elevated relative to non-NGF-treated cells. Partial microsequencing of this material reveals 2 internal sequences that are identical to a 14-residue sequence encoded by the NGF-regulated clone 73 mRNA, but not to sequences of other known proteins. An antiserum raised against a 19-residue synthetic peptide corresponding to the deduced C-terminus of the protein encoded by the NGF-regulated clone 73 mRNA specifically recognizes the 58,000-Mr protein. Properties of the 58-kilodalton protein strongly suggest that it corresponds to an intermediate filament protein (peripherin) previously identified in PC12 cells and in peripheral and certain CNS neurons. Identification of the intermediate filament protein encoded by an NGF-induced message should facilitate studies of its regulation and function.     

Chromogranin, an integral membrane protein

Chromogranin is the major soluble protein of the adrenal medulla chromaffin granule and is secreted upon nervous stimulation. Using antisera to pure chromogranin in immunoblotting procedures, we show that chromogranin is the major integral membrane protein as well. Extraction of chromaffin granule membranes with low salt, high salt, chelating agents, or calcium-containing solutions does not remove the chromogranin from the membranes. The membrane form of chromogranin can be purified on a C-18 semi-preparative column using high pressure liquid chromatography. Amino-terminal sequence data indicate that the membrane and soluble forms of chromogranin are identical or very similar.     

StarD7 behaves as a fusogenic protein in model and cell membrane bilayers

StarD7 is a surface active protein, structurally related with the START lipid transport family. So, the present work was aimed at elucidating a potential mechanism of action for StarD7 that could be related to its interaction with a lipid-membrane interface. We applied an assay based on the fluorescence de-quenching of BD-HPC-labeled DMPC-DMPS 4:1 mol/mol SUVs (donor liposomes) induced by the dilution with non-labeled DMPC-DMPS 4:1 mol/mol LUVs (acceptor liposomes). Recombinant StarD7 accelerated the dilution of BD-HPC in a concentration-dependent manner. This result could have been explained by either a bilayer fusion or monomeric transport of the labeled lipid between donor and acceptor liposomes. Further experiments (fluorescence energy transfer between DPH-HPC/BD-HPC, liposome size distribution analysis by dynamic light scattering, and the multinuclear giant cell formation induced by recombinant StarD7) strongly indicated that bilayer fusion was the mechanism responsible for the StarD7-induced lipid dilution. The efficiency of lipid dilution was dependent on StarD7 electrostatic interactions with the lipid-water interface, as shown by the pH- and salt-induced modulation. Moreover, this process was favored by phosphatidylethanolamine which is known to stabilize non-lamellar phases considered as intermediary in the fusion process. Altogether these findings allow postulate StarD7 as a fusogenic protein.