A live cell, image-based approach to understanding the enzymology and pharmacology of 2-bromopalmitate and palmitoylation.
The addition of a lipid moiety to a protein increases its hydrophobicity and subsequently its attraction to lipophilic environments like membranes. Indeed most lipid-modified proteins are localized to membranes where they associate with multiprotein signaling complexes. Acylation and prenylation are the two common categories of lipidation. The enzymology and pharmacology of prenylation are well understood but relatively very little is known about palmitoylation, the most common form of acylation. One distinguishing characteristic of palmitoylation is that it is a dynamic modification. To understand more about how palmitoylation is regulated, we fused palmitoylation substrates to fluorescent proteins and reported their subcellular distribution and trafficking. We used automated high-throughput fluorescence microscopy and a specialized computer algorithm to image and measure the fraction of palmitoylation reporter on the plasma membrane versus the cytoplasm. Using this system we determined the residence half-life of palmitate on the dipalmitoyl substrate peptide from GAP43 as well as the EC(50) for 2-bromopalmitate, a common inhibitor of palmitoylation.
Mikic
Ivana
I
Vala Sciences Inc., La Jolla, CA, USA.
Planey
Sonia
S
Zhang
Jun
J
Ceballos
Carolina
C
Seron
Terri
T
von Massenbach
Benedikt
B
Watson
Rachael
R
Callaway
Scott
S
McDonough
Patrick M
PM
Price
Jeffrey H
JH
Hunter
Edward
E
Zacharias
David
D
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D020128:Inhibitory Concentration 50; D008055:Lipids / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010168:Palmitates / Q000737:chemistry* / Q000494:pharmacology*; D019308:Palmitic Acid / Q000737:chemistry* / Q000494:pharmacology*; D015398:Signal Transduction
D001426:Bacterial Proteins; D008055:Lipids; D008164:Luminescent Proteins; D010168:Palmitates; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; C022776:2-bromopalmitate; D019308:Palmitic Acid; D003545:Cysteine