Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase.
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.
Institute of Chemical Biology, Department of Chemistry, Imperial College London, South Kensington SW7 2AZ, UK.
Thomas
T
Lanyon-Hogg
Institute of Chemical Biology, Department of Chemistry, Imperial College London, South Kensington SW7 2AZ, UK.
Naoko
N
Masumoto
Institute of Chemical Biology, Department of Chemistry, Imperial College London, South Kensington SW7 2AZ, UK.
George
G
Bodakh
Molecular Medicine Section, National Heart & Lung Institute, Imperial College London, South Kensington SW7 2AZ, UK.
Antonio D
AD
Konitsiotis
Institute of Chemical Biology, Department of Chemistry, Imperial College London, South Kensington SW7 2AZ, UK.
Emmanuelle
E
Thinon
Molecular Medicine Section, National Heart & Lung Institute, Imperial College London, South Kensington SW7 2AZ, UK.
Ursula R
UR
Rodgers
Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK; OPPF-UK, The Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell, Oxfordshire, OX11 0FA, UK.
Raymond J
RJ
Owens
Molecular Medicine Section, National Heart & Lung Institute, Imperial College London, South Kensington SW7 2AZ, UK. Electronic address: t.magee@imperial.ac.uk.
Anthony I
AI
Magee
Institute of Chemical Biology, Department of Chemistry, Imperial College London, South Kensington SW7 2AZ, UK. Electronic address: e.tate@imperial.ac.uk.
Edward W
EW
Tate
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019921:Biotinylation; D057930:Click Chemistry; D003902:Detergents / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D057809:HEK293 Cells; D053823:Hedgehog Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D054878:Lipoylation / Q000187:drug effects; D008320:Maltose / Q000031:analogs & derivatives / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010171:Palmitoyl Coenzyme A / Q000031:analogs & derivatives / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D003902:Detergents; D004791:Enzyme Inhibitors; D005231:Fatty Acids, Unsaturated; D053823:Hedgehog Proteins; D055767:Immobilized Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C509239:SHH protein, human; C411362:dodecyl maltopyranoside; D010171:Palmitoyl Coenzyme A; D008320:Maltose; D019809:Streptavidin; D000217:Acyltransferases; C105599:HHAT protein, human
Click chemistry; Hedgehog acyltransferase; MBOAT; Protein palmitoylation