ArticleAnalytical biochemistry2015
Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase.
Article in Analytical biochemistry, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed, 27 citations in OpenAlex.
- Design, Synthesis, and Evaluation of Inhibitors of Hedgehog Acyltransferase.Journal of medicinal chemistry · 2024Article
- A rising tide lifts all MBOATs: recent progress in structural and functional understanding of membrane boundFrontiers in physiology · 2023Review
- A Direct in vitro Fatty Acylation Assay for Hedgehog Acyltransferase.Bio-protocol · 2022Article
- Review
- A Not-So-Ancient Grease History: Click Chemistry and Protein Lipid Modifications.Chemical reviews · 2021Review
- Palmitoylation of Hedgehog proteins by Hedgehog acyltransferase: roles in signalling and disease.Open biology · 2021Review
- Acylation-coupled lipophilic induction of polarisation (Acyl-cLIP): a universal assay for lipid transferase and hydrolase enzymes.Chemical science · 2019Article
- cat-ELCCA: catalyzing drug discovery through click chemistry.Chemical communications (Cambridge, England) · 2018Article
- Microfluidic Mobility Shift Assay for Real-Time Analysis of Peptide N-Palmitoylation.SLAS discovery : advancing life sciences R & D · 2017Article
- Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods.Biochemistry and biophysics reports · 2017Article
- Characterization of Hedgehog Acyltransferase Inhibitors Identifies a Small Molecule Probe for Hedgehog Signaling by Cancer Cells.ACS chemical biology · 2016Article
- Highly sensitive detection of lipopolysaccharides using an aptasensor based on hybridization chain reaction.Scientific reports · 2016Article
- Synthesis and characterisation of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine inhibitors of Hedgehog acyltransferase.Data in brief · 2016Article
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Authors and funding
9 authors at 3 institutions in 2 countries.
Funding
Abstract
Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click-ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click-ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click-ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.