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William J Driscoll

Publications and source records attributed to William J Driscoll.

6 recordsLinked to original sources
PubMed2006

In vivo evidence that N-oleoylglycine acts independently of its conversion to oleamide.

Oleamide (cis-9-octadecenamide) is a member of an emerging class of lipid-signaling molecules, the primary fatty acid amides. A growing body of evidence indicates that oleamide mediates fundamental neurochemical processes including sleep, thermoregulation, and nociception. Nevertheless, the mechanism for oleamide biosynthesis remains unknown. The leading hypothesis holds that oleamide is synthesized from oleoylglycine via the actions of the peptide amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The present study investigated this hypothesis using pharmacologic treatments, physiologic assessments, and measurements of serum oleamide levels using a newly developed enzyme-linked immunosorbant assay (ELISA). Oleamide and oleoylglycine both induced profound hypothermia and decreased locomotion, over equivalent dose ranges and time courses, whereas, closely related compounds, stearamide and oleic acid, were essentially without effect. While the biologic actions of oleamide and oleoylglycine were equivalent, the two compounds differed dramatically with respect to their effects on serum levels of oleamide. Oleamide administration (80mg/kg) elevated blood-borne oleamide by eight-fold, whereas, the same dose of oleoylglycine had no effect on circulating oleamide levels. In addition, pretreatment with the established PAM inhibitor, disulfiram, produced modest reductions in the hypothermic responses to both oleoylglycine and oleamide, suggesting that the effects of disulfiram were not mediated through inhibition of PAM and a resulting decrease in the formation of oleamide from oleoylglycine. Collectively, these findings raise the possibilities that: (1) oleoylglycine possesses biologic activity that is independent of its conversion to oleamide and (2) the increased availability of oleoylglycine as a potential substrate does not drive the biosynthesis of oleamide.

Animals↗
PubMed2006

De novo biosynthetic profiling of high abundance proteins in cystic fibrosis lung epithelial cells.

In previous studies with cystic fibrosis (CF) IB3-1 lung epithelial cells in culture, we identified 194 unique high abundance proteins by conventional two-dimensional gel electrophoresis and mass spectrometry (Pollard, H. B., Ji, X.-D., Jozwik, C. J., and Jacobowitz, D. M. (2005) High abundance protein profiling of cystic fibrosis lung epithelial cells. Proteomics 5, 2210-2226). In the present work we compared the IB3-1 cells with IB3-1/S9 daughter cells repaired by gene transfer with AAV-(wild type)CFTR. We report that gene transfer resulted in significant changes in silver stain intensity of only 20 of the 194 proteins. However, simultaneous measurement of de novo biosynthetic rates with [(35)S]methionine of all 194 proteins in both cell types resulted in the identification of an additional 31 CF-specific proteins. Of the 51 proteins identified by this hybrid approach, only six proteins changed similarly in both the mass and kinetics categories. This kinetic portion of the high abundance CF proteome, hidden from direct analysis of abundance, included proteins from transcription and signaling pathways such as NFkappaB, chaperones such as HSC70, cytoskeletal proteins, and others. Connectivity analysis indicated that approximately 30% of the 51-member hybrid high abundance CF proteome interacts with the NFkappaB signaling pathway. In conclusion, measurement of biosynthetic rates on a global scale can be used to identify disease-specific differences within the high abundance cystic fibrosis proteome. Most of these kinetically defined proteins are unaffected in expression level when using conventional silver stain analysis. We anticipate that this novel hybrid approach to discovery of the high abundance CF proteome will find general application to other proteomic problems in biology and medicine.

Cell Line↗
PubMed2005

Murine atrial HL-1 cells express highly active peptidylglycine alpha-amidating enzyme.

Peptidylglycine-alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) catalyzes the rate limiting step in peptide alpha-amidation, a posttranslational modification that is essential for receptor recognition and signal transduction. Secretory granules of the cardiac atrium contain the highest natural concentration of PHM and clearly demonstrate regulation of PHM expression and activity. The HL-1 atrial myocyte cell line faithfully maintains the differentiated phenotype of native atrial cells and thus provides an in vitro model system for investigating the mechanisms that regulate PHM. We observed that the specific activity of PHM expressed in HL-1 cells is five times higher than that found in rat atrium. The increased activity of HL-1 cell PHM was not reflected by a difference in Km for peptide substrate, change in copper optimum, altered sensitivity to inactivation by suicide inhibitor or variance in response to limited proteolysis by trypsin. Additionally, mixing experiments indicated that the increased activity in HL-1 cells versus rat atrium was not due to a diffusible factor. Based upon these findings we propose that the increased Vmax of HL-1 cell PHM results from a structural or conformational difference that involves either differential posttranslational modification and/or a high affinity chaperone that serves to regulate enzymatic activity by protein-protein interaction. The mechanism involved may participate in physiologic regulation of PHM.

Animals↗
PubMed2004

Proteomic analysis of rat atrial secretory granules: a platform for testable hypotheses.

The recent development of powerful proteomic tools has enabled investigators to directly examine the population of proteins present in defined biological systems. We report here the first proteomic analysis of atrial secretory granules. Approximately 100 distinct protein components of the atrial secretory granule proteome were detected using subcellular fractionation and one-dimensional SDS-PAGE in conjunction with peptide mass fingerprinting by MALDI-TOF mass spectrometry. Of this number, 61 proteins were clearly identified by high probability data matches and repeated observation. The majority of the proteome was found to be membrane-associated with the most prominent proteins being peptidylglycine alpha-amidating monooxygenase (PAM) and pro-atrial natriuretic peptide (pro-ANP). This proteomic analysis of the rat atrium secretory granule produced an assembly of proteins with a diverse array of reported functions. The identified proteins fall into seven functional categories: (1) granular transport, docking and fusion; (2) signal transduction; (3) calcium-binding/calcium-dependent; (4) cellular architecture/chaperoning; (5) peptide/protein processing; (6) hormone; (7) proton transport. The novel finding of several protein processing enzymes and signal transduction proteins offer new perspectives on how pro-ANP is stored and processed to ANP during release. Accordingly, defining the proteome of the atrial secretory granule provides a framework for the development of new hypotheses that address key mechanisms governing granule function and ANP secretion.

Animals↗
PubMed2003

Peptidylglycine-alpha-amidating monooxygenase and pro-atrial natriuretic peptide constitute the major membrane-associated proteins of rat atrial secretory granules.

Peptidylglycine-alpha-amidating monooxygenase (PAM) is a bi-functional enzyme known to catalyze the post-translational bioactivation of signaling peptides. Although PAM is highly concentrated within the cardiac atrium, this tissue does not produce appreciable amounts of alpha-amidated peptides and thus, the function of PAM in atrium remains largely unknown. In this study, we demonstrate that PAM co-localizes in atrial secretory granules with the storage form of atrial natriuretic peptide (pro-ANP, amino acids 1-126), a hormone involved in the maintenance of blood pressure and fluid homeostasis. ANP is not amidated by PAM, but rather is processed to its active form (amino acids 99-126) by the proteolytic cleavage of pro-ANP. We demonstrate here by subcellular fractionation and biochemical analyses that PAM co-localizes with pro-ANP in secretory granules, where together they constitute the two most abundant membrane-associated proteins, accounting for approximately 95% of the total granular membrane protein. Respectively, light and electron microscopic immunohistochemistry show intense staining for PAM in atrial cardiomyocyctes and subcellular localization of PAM to secretory granules. Additionally, we demonstrate that while pro-ANP is readily found in the soluble contents of the granule lumen, significant amounts remain tightly associated with the membranes even after vigorous washing and estimate the molar ratio of pro-ANP to PAM to be approximately 30:1 in the membrane fraction. We postulate here that the primary function of PAM in the atrium is structural rather than enzymatic. In this regard, PAM may contribute to the packaging of pro-ANP within the secretory granule and possibly function in the presentation of pro-ANP for proteolytic processing.

Animals↗