**1. Introduction**

Palmitoylation is a reversible, post-translational modification of a protein through the addi‐ tion of the 16-carbon fatty acid, palmitate, to a cysteine residue. There are two types of pal‐ mitoylation, one called thio- or *S*-palmitoylation in which palmitate is added to the thiol side chain of a cysteine residue via a labile thioester bond [1]. The other type, *N*-palmitoyla‐ tion, is the addition of palmitate to an N-terminal cysteine via a stable amide bond [2]. The two forms of palmitoylation are regulated by different families of palmitoyl acyltransferases (PATs)—*S*-palmitoylation via a family of multi-pass transmembrane proteins called DHHC (Asp-His-His-Cys) proteins [3] and *N*-palmitoylation via a family of multi-pass transmem‐ brane proteins termed membrane-bound *O*-acyltransferase [4]. S-palmitoylation, the focus of this chapter, is more common and because of the labile thioester bond, can dynamically regulate protein sorting and function.

Palmitoylation increases the lipophilicity of the modified protein often changing its subcel‐ lular distribution in both dramatic and subtle ways. The larger-scale changes occur when cy‐ toplasmic proteins relocate from the cytoplasm to membrane and when integral membrane proteins move from one membrane system to another, such as from the endoplasmic reticu‐ lum (ER) to the plasma membrane (PM). The more subtle changes, in terms of distance, oc‐ cur at the nanoscale level within a membrane. The increase in lipophilicity upon palmitoylation often results in an altered affinity for a particular lipid microenvironment within that membrane [5]. For example, lipid rafts are small islands in membranes with dis‐ tinct lipid compositions that selectively attract or exclude both peripheral (often exclusively by virtue of palmitoylation) and integral membrane palmitoylated proteins. Palmitoylated proteins have affinity for lipid rafts that are rich in cholesterol, while prenylated proteins have little or no affinity for these rafts [5]. Such lipophilicity-driven changes in protein dis‐

© 2013 Planey; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

tribution may alter access of a palmitoylated protein to extracellular ligands (when the pro‐ tein moves from the ER to the PM), protein-protein interactions, or the engagement of the palmitoyl-protein in multi-molecular signaling complexes. The role of palmitoylation as a versatile protein sorting signal, regulating intracellular protein trafficking and targeting to membrane microdomains has been reviewed recently [6]. Palmitate may be the most com‐ mon lipid species to occupy cysteine residues, but it is not the only one. Marilyn Resh and colleagues identified the lipid moieties resident on the cysteine residue of the N-terminal tail of Src family kinases [7-9]. While for these proteins the cysteine residue near the N-terminus is most frequently palmitoylated, it is also modified by palmitoleate, stearate, or oleate with a frequency that is apparently related to the abundance of palmitate in cells [10]. The phys‐ iological differences that result from proteins being modified by these other lipids has not been explored extensively; however, given their different physical properties, it seems rea‐ sonable that their impact on a protein should be subtly different than palmitate.

teins remained a mystery and somewhat controversial until only recently. The apparent ab‐ sence of a consensus site for palmitoylation encoded by the sequence of amino acid residues surrounding palmitoyl cysteines, as well as the difficulty in purifying and identifying the enzymes capable of mediating the reaction, led many to believe that it was autocatalytic. Given these issues and the high reactivity of cysteines and palmitoyl-CoA, especially in *in vitro* protein palmitoylation assays, the possibility was not unreasonable [11, 14, 15]. Many of the arguments for and against autocatalytic palmitoylation have been reviewed recently [16].Yet, given the prevalence of palmitoylated proteins in parts of cells where signaling events are so highly concentrated, complex, and regulated, such as the neuronal synapse, it seemed somewhat unreasonable that all regulation of palmitoylation could be left to diffu‐ sion—a nagging reality that kept the search for an enzymatic mechanism alive despite the arguments to the contrary. Additionally, there was evidence over the years in support of the idea that these enzymes existed because PAT activity in detergent solubilized protein frac‐ tions had been measured using viral glycoproteins [17], p59*fyn* [18], and H-Ras [19] as sub‐

Discovery of Selective and Potent Inhibitors of Palmitoylation

http://dx.doi.org/10.5772/52503

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The experiments that conclusively provided the molecular identity of PATs were presented in a series of papers spanning almost a decade. The experimental model organism that ultimate‐ ly provided the information was yeast. First, palmitoylation-dependent alleles of yeast *RAS2* were identified. A genetic screen designed to identify mutations that rendered cells non-via‐ ble if Ras2p was not palmitoylated was utilized to identify mutations in two genes- *ERF2* and *ERF4/SHR*5 [20, 21]. These mutations resulted in diminished palmitoylation of Ras2p and mis‐ localization of GFP-Ras2p (respectively or it takes both mutations to cause both effects [20, 22]). However, it could not be decisively concluded if the mutations in *ERF2* and *ERF4* were affect‐ ing Ras2p palmitoylation directly or indirectly by altering Ras2p trafficking (which could have

In collaboration with Maurine Linder, Deschenes and colleagues used an *in vitro* palmitoyla‐ tion assay to show that Erf2p and Erf4p together constituted a Ras2p PAT that used palmito‐ yl-CoA as a donor [23]. Erf2p is a ~42-kDa integral membrane protein that is expressed in the ER. The protein contains the DHHC-CRD (Asp-His-His-Cys-cysteine rich domain), also referred to as the NEW1 or zf-DHHC domain (PF01529), which is found in an extensive fam‐ ily of membrane proteins ranging from unicellular eukaryotes to humans [24, 25]. This do‐ main is now recognized as the molecular signature for PATs that add palmitate to cysteines

At almost the same time that the Erf2p/Erf4p complex was identified as the Ras2p PAT, Akr1p was identified as a PAT with specificity for Yck2p [26]. An important clue leading to the relationship between these two proteins came from the fact that mutants in both Ras2p and Yck2P exhibited a reduced rate of pheromone receptor internalization [27, 28]. Akr1p contains a DHYC-CRD instead of a DHHC-CRD as well as ankyrin repeats not present in Erf2p. The DHYC motif present in three yeast proteins (Akr1p, Akr2p and Pfa5) does not appear to occur in the mammalian genome. Akr1p and Akr2p are most closely related to the mammalian HIP14 (DHHC17) and HIP14L (DHHC13) which contains the variant DQHC—

prevented an interaction between the palmitoyl acyltransferase and Ras2p).

the only observed mammalian deviation from DHHC [3].

strates among others.

via a labile thioester bond.

Unlike other forms of lipidation such as myristoylation and prenylation, palmitoylation is reversible, by virtue of the labile thioester bond. This allows for dynamic regulation of the protein's lipophilicity [11-13]. By contrast, prenyl groups are attached to cysteines by a sta‐ ble thioether bond and myristate to glycines by a stable amide bond. It is now apparent that many instances of palmitoylation are enzymatically mediated by a family of palmitoyl acyl‐ transferases (PATs), whereas the mechanisms for depalmitoylation are poorly understood. Nevertheless, it is known that palmitate cycles on and off of many proteins at variable rates ranging from minutes to days. Such dynamic regulation makes palmitoylation unique among post-translational protein lipid modifications and places it in a category similar to phosphorylation. Discovering the molecular identity of PATs was a pivotal event that dra‐ matically accelerated the pace of discovery in the field. Likewise, there has been increased interest in palmitoylation partly because many of the genes encoding PATs have been linked to human diseases like cancer. With a greater understanding of how palmitate is en‐ zymatically attached to proteins, some of the most interesting questions include: What are the substrate(s) of each PAT?; how does a PAT recognize and palmitoylated a substrate?; how are PATs regulated?; and how is depalmitoylation regulated? The answers to these questions are beginning to unfold due to the recent discovery of pharmacological modula‐ tors of palmitoylation as well as the development of novel assays and refinement of existing assays. Our ability to understand palmitoylation and its importance to human health and disease is only as good as the methods we use to test our hypotheses. Thus, the discovery of potent and selective inhibitors of palmitoylation as well as the continued development of as‐ says with increased sensitivity and selectivity is critical to this venture.
