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Review
. 2006 Oct;40(2):166-70.
doi: 10.1016/j.ymeth.2006.06.019.

In vitro and cellular assays for palmitoyl acyltransferases using fluorescent lipidated peptides

Charles E Ducker  1 , Jeremiah M Draper, Zuping Xia, Charles D Smith
Affiliations
Review

In vitro and cellular assays for palmitoyl acyltransferases using fluorescent lipidated peptides

Charles E Ducker et al. Methods. 2006 Oct.

Abstract

Protein palmitoylation is emerging as an important post-translational modification in development as well as in the establishment and progression of diseases such as cancer. This chapter describes the use of fluorescent lipidated peptides to characterize palmitoyl acyltransferase (PAT) activities in vitro and in intact cells. The peptides mimic two motifs that are enzymatically palmitoylated, i.e. C-terminal farnesyl and N-terminal myristoyl sequences. These substrate peptides can be separated from the palmitoylated product peptides by reversed-phase HPLC, detected and quantified by the fluorescence of their NBD label. Through these methods, the activities of PATs toward these alternate substrates in isolated membranes or intact cells can be quantified. The in vitro assay has been used to characterize human PATs and to identify inhibitors of these enzymes. The cellular assay has been useful in elucidating the kinetics of protein palmitoylation by PATs in situ, and the sub-cellular distribution of the palmitoylated products.

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Figures

Fig. 1
Fig. 1
Structures of deprotected PAT substrate peptides. (NBD)-CLC(OMe)-Farn contains the C-terminal farnesylated cysteine and an upstream palmitoylatable cysteine found in proteins such as N- and H-Ras and recognized by Type I PATs. Myr-GC(NBD) mimics proteins with an N-terminal myristoylated glycine followed by a palmitoylatable cysteine residue, such as Src-related tyrosine kinases, and is recognized by Type 2 PATs.
Fig. 2
Fig. 2
HLPC separation of substrate peptides and palmitoylated product peptides. Chromatograms from (NBD)-CLC(OMe)-Farn and Myr-GC-(NBD) peptides (1 µM) treated with 2 µM palmitoyl-CoA at pH 8.2 for 15 min at 37 °C to induce chemical palmitoylation are shown. The peptides were extracted and subjected to HPLC analysis as described in the text. Peaks were verified as non-palmitoylated (a) and palmitoylated (b) by mass spectroscopy.
Fig. 3
Fig. 3
Subcellular localization of palmitoylated peptides. Myr-GC-(NBD) (left panel) or (NBD)-CLC(OMe)-Farn (right panel) at a final concentration of 1 µM was incubated with SKOV3 cells for 15 min. The media was removed and the cells were rapidly washed twice with cold PBS and imaged using a Leica DMIRE2 confocal microscope with Leica confocal software.
Fig. 4
Fig. 4
Representative PAT inhibitors identified by screening. Structures of selected compounds identified as PAT inhibitors are shown. Inhibition of PAT activity was determined using in vitro palmitoylation assays with either the (NBD)-CLC(OMe)-Farn (open bars) or Myr-GC-(NBD) (filled bars) peptides. Each compound was tested at a concentration of 25 µg/ml. See [22] for further details.
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