cat‐ELCCA: A Robust Method To Monitor the Fatty Acid Acyltransferase Activity of Ghrelin <i>O</i>‐Acyltransferase (GOAT)

Garner, Amanda L.; Janda, Kim D.

doi:10.1002/ange.201003387

Cited by 11 publications

(16 citation statements)

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“…The merging of chemistry and biology for assay design and development is a unique approach for solving challenging problems, such as the discovery of RNA-specific ligands. As we have already demonstrated the potential of cat-ELCCA for monitoring and inhibiting post-translational modifications (e.g., fatty acid acylation) 24,25 and now RNA, we believe that cat-ELCCA has the potential for translation to other difficult biological targets, including protein−protein and RNA−protein interactions. Future efforts will be devoted toward these goals.…”

Section: ■ Results and Discussionmentioning

confidence: 96%

“…24 The key step is the use of copper(I)-catalyzed alkyne− azide cycloaddition chemistry to "click" an azido-horseradish peroxidase (HRP) to an alkynyl biomolecule for enzymemediated signal amplification. In addition to catalyzed signal turnover, a significant advantage of cat-ELCCA with respect to ligand screening is the fact that this assay approach is not subject to compound interference like traditional fluorescence assays.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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High-Throughput Platform Assay Technology for the Discovery of pre-microRNA-Selective Small Molecule Probes

2014

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MicroRNAs (miRNA) play critical roles in human development and disease. As such, the targeting of miRNAs is considered attractive as a novel therapeutic strategy. A major bottleneck toward this goal, however, has been the identification of small molecule probes that are specific for select RNAs and methods that will facilitate such discovery efforts. Using pre-microRNAs as proof-of-concept, herein we report a conceptually new and innovative approach for assaying RNA-small molecule interactions. Through this platform assay technology, which we term catalytic enzyme-linked click chemistry assay or cat-ELCCA, we have designed a method that can be implemented in high throughput, is virtually free of false readouts, and is general for all nucleic acids. Through cat-ELCCA, we envision the discovery of selective small molecule ligands for disease-relevant miRNAs to promote the field of RNA-targeted drug discovery and further our understanding of the role of miRNAs in cellular biology.

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Section: ■ Results and Discussionmentioning

confidence: 96%

Section: ■ Results and Discussionmentioning

confidence: 99%

High-Throughput Platform Assay Technology for the Discovery of pre-microRNA-Selective Small Molecule Probes

2014

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“…The observed reduction in gastric and plasmatic GOAT protein may have a bearing with the parallel 67% decrease in circulating acyl ghrelin observed 2 h after abdominal surgery. However, this will need to be ascertained also at the level of GOAT enzyme activity by use of enzymelinked click-chemistry (cat-ELCCA), which was recently developed (25). Furthermore, in addition to reduced GOAT protein expression, it cannot be ruled out that other mechanisms participating in the secretion rate of acyl ghrelin or changes in the activity of other enzymes, namely hepatic esterases involved in deacylation (19) may also contribute to the observed altered ratio of acyl to desacyl ghrelin.…”

Section: Discussionmentioning

confidence: 99%

Abdominal surgery inhibits circulating acyl ghrelin and ghrelin-O-acyltransferase levels in rats: role of the somatostatin receptor subtype 2

Stengel

Goebel-Stengel

Wang

et al. 2011

American Journal of Physiology-Gastrointestinal and Liver Physi

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NWG, Rivier J, Taché Y. Abdominal surgery inhibits circulating acyl ghrelin and ghrelin-O-acyltransferase levels in rats: role of the somatostatin receptor subtype 2. Am J Physiol Gastrointest Liver Physiol 301: G239 -G248, 2011. First published June 2, 2011; doi:10.1152/ajpgi.00018.2011.-Clinical studies are evaluating the efficacy of synthetic ghrelin agonists in postoperative ileus management. However, the control of ghrelin secretion under conditions of postoperative gastric ileus is largely unknown. Peripheral somatostatin inhibits ghrelin secretion in animals and humans. We investigated the time course of ghrelin changes postsurgery in fasted rats and whether somatostatin receptor subtype 2 (sst2) signaling is involved. Abdominal surgery (laparotomy and 1-min cecal palpation) induced a rapid and long-lasting decrease in plasma acyl ghrelin levels as shown by the 64, 67, and 59% reduction at 0.5, 2, and 5 h postsurgery, respectively, compared with sham (anesthesia alone for 10 min, P Ͻ 0.05). Levels were partly recovered at 7 h and fully restored at 24 h. The percentage of acyl ghrelin reduction was significantly higher than that of desacyl ghrelin at 2 h postsurgery and not at any other time point. This was associated with a 48 and 23% decrease in gastric and plasma ghrelin-O-acyltransferase protein concentrations, respectively (P Ͻ 0.001). Ghrelin-positive cells in the oxyntic mucosa expressed sst2a receptor and the sst2 agonist S-346-011 inhibited fasting acyl ghrelin levels by 64 and 77% at 0.5 and 2 h, respectively. The sst2 antagonist S-406-028 prevented the abdominal surgery-induced decreased circulating acyl ghrelin but not the delayed gastric emptying assessed 0.5 h postinjection. These data show that activation of sst2 receptor located on gastric X/A-like cells plays a key role in the rapid inhibition of circulating acyl ghrelin induced by abdominal surgery while not being primarily involved in the early phase of postoperative gastric ileus. gastric emptying; somatostatin receptor 2 agonist; somatostatin receptor 2 antagonist; X/A-like cell POSTOPERATIVE GASTRIC ILEUS is a condition that develops as a consequence of surgery and is associated with delayed gastric emptying and intestinal transit (41). Since prolonged postoperative ileus (POI) is an iatrogenic condition leading to prolonged hospitalization resulting in increased health care costs, the development of effective management strategies to prevent POI is clinically important (74). Recently, the food intake stimulating and gastroprokinetic effects of the peptide hormone ghrelin is gaining recognition for the treatment of POI (9). The long-acting synthetic agonist Tranzyme Pharma-101 was reported to accelerate the recovery of the upper and lower gastrointestinal tract in patients undergoing major abdominal surgery (47), consistent with preclinical evidence that peripheral administration of ghrelin agonists reversed the abdominal surgery-induced delayed gastric emptying in rats and dogs (69). However, the regulation of ghrelin under condition...

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“…Addition of long-chain fatty acyl-CoA increases the activity of GOAT in an in vitro assay system containing the cell membrane fraction, presumably by inhibiting the degradation of octanoyl CoA [26,27]. It is possible that the presence of sufficient levels of long-chain fatty acyl-CoA contributed to the preservation of octanoyl-CoA, either incorporated from the circulation or derived from the b-oxidation of long-chain fatty acids.…”

Section: Discussionmentioning

confidence: 99%

“…To measure ghrelin concentrations in culture medium, the collected culture media were centrifuged, the resulting supernatants were immediately applied to Sep-Pak C18 cartridges and processed as described above. Two types of ghrelin RIA were performed: C-RIA, in which an anti-COOH-terminal ghrelin (amino acids [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] antiserum was used to detect both ghrelin and des-acyl ghrelin (total ghrelin); and N-RIA, in which an anti-NH 2 -terminal ghrelin (amino acids 1-11) antiserum was used to detect (acylated) ghrelin only, as previously described [2,17].…”

Section: Measurement Of Ghrelin Concentrations In Cells and Mediummentioning

confidence: 99%

High incorporation of long‐chain fatty acids contributes to the efficient production of acylated ghrelin in ghrelin‐producing cells

et al. 2016

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Edited by L aszl o NagyThe mechanisms for supplying octanoic acid for ghrelin acylation in X/A-like cells are incompletely understood. We found that long-chain fatty acids were incorporated at a higher rate in the ghrelin-producing cell line MGN3-1 than in MIN6 cells, in part due to higher expression level of long-chain fatty acylCoA synthetase family member 1 (Acsl1). Inhibition of ACSLs by triacsin C profoundly suppressed acylated ghrelin production. These results suggest that high incorporation of long-chain fatty acids into the ghrelin-producing cells plays a role in the supply of octanoic acid for ghrelin acylation.

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cat‐ELCCA: A Robust Method To Monitor the Fatty Acid Acyltransferase Activity of Ghrelin O‐Acyltransferase (GOAT)

Cited by 11 publications

References 56 publications

High-Throughput Platform Assay Technology for the Discovery of pre-microRNA-Selective Small Molecule Probes

High-Throughput Platform Assay Technology for the Discovery of pre-microRNA-Selective Small Molecule Probes

Abdominal surgery inhibits circulating acyl ghrelin and ghrelin-O-acyltransferase levels in rats: role of the somatostatin receptor subtype 2

High incorporation of long‐chain fatty acids contributes to the efficient production of acylated ghrelin in ghrelin‐producing cells

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