**2. Palmitoylation and DHHC proteins**

#### **2.1. Molecular identity of palmitoyl acyltransferases (PATs)**

It has been known for many years that palmitoylation is a critical regulator of diverse and complex signaling networks, but the mechanism responsible for palmitoylation of most pro‐ 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‐ strates among others.

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‐

252 Drug Discovery

sonable that their impact on a protein should be subtly different than palmitate.

says with increased sensitivity and selectivity is critical to this venture.

It has been known for many years that palmitoylation is a critical regulator of diverse and complex signaling networks, but the mechanism responsible for palmitoylation of most pro‐

**2. Palmitoylation and DHHC proteins**

**2.1. Molecular identity of palmitoyl acyltransferases (PATs)**

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‐

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 prevented an interaction between the palmitoyl acyltransferase and Ras2p).

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 via a labile thioester bond.

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 the only observed mammalian deviation from DHHC [3].

### **2.2. The ZDHHC family of PATs**

The mammalian genome contains at least 23 members of the *ZDHHC* PAT gene family iden‐ tified by the presence of the signature DHHC-cysteine rich domain. Members of the family had been identified as being genes of interest (e.g., "REAM" in metastatic cancer [29]) prior to understanding their function. The genomic structure of *ZDHHC* genes varies widely, in‐ cluding the number and differential use of exons that are spliced together to generate the mRNA. EC gene analyses (http://genome.ewha.ac.kr/ECgene/) of the mRNAs that encode PATs suggest that all of the genes are alternatively spliced at various sites throughout the protein coding sequence as well as within untranslated regions. Many of the putative, alter‐ natively-spliced exons are predicted to encode small peptides that change the structure of the protein in a way that may alter substrate specificity. Similarly, splicing may alter sites for other post-translational modifications, such as phosphorylation or glycosylation, all of which may regulate activity, substrate specificity, subcellular distribution, or interactions with non-substrate proteins. *ZDHHC7*, for example, alters the use of a 111 bp exon that is differentially and specifically expressed in tissues such as placenta, lung, liver, thymus, and small intestine [30]. This exon encodes a 37-residue peptide (EKSSDCRP‐ SACTVKTGLDPTLVGICGEGTESVQSLLL) within the intracellular loop between trans‐ membrane domain 2 (TM2) and TM3 that contains a PKC phosphorylation site. It is conceivable that phosphorylation of this serine changes DHHC7 in such a way that sub‐ strate specificity or the rate of palmitate transfer activity is altered. In addition to alternative mRNA splicing, aberrant splicing induced by mutations or single nucleotide polymor‐ phisms has been shown to occur in at least two ZDHHC genes. A splice-site mutation in highly conserved residues of *ZDHHC9*, a PAT that has been shown to palmitoylate H-Ras and N-Ras [31], has been described in families with X-linked mental retardation (XLMR) [32]. This mutation creates an additional, stronger splice-donor site 140 nt before (toward the 5' end) the normal donor site. Usage of the new site results in a mRNA that is frameshifted and that encodes a truncated protein. Single nucleotide polymorphisms that affect splicing of *ZDHHC8* have also been implicated in schizophrenia [33] (also see: [34-36]).

seems somewhat unlikely they would do so in this arrangement as it represents a state in which it would be difficult to perform these functions. The highest-scoring predictions of the membrane topology using TMpred show that the human protein sequence of DHHC11 and -16 should contain four TM domains, DHHC13 eight TM domains, and DHHC22 either four or five TM domains, with each model placing the DHHC-CRD motif inside the cells. There is clear disparity among the predictions generated by the algorithms available and ul‐ timately, any of these predictions of topology must be confirmed or disproved by experi‐ mental data. In any case, for a member of the PAT family to function as a PAT, the DHHC-CRD motif should probably reside in the cytoplasm (Figure 1A). The regions of the PAT proteins that contain the greatest diversity at the amino-acid level are the N- and C-terminal

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255

**DHHC-CRD**

**DPG TTxE**

**TM1 TM2 TM3 TM4**

**Figure 1.** Predicted general structure of PATs. A) The predicted topology of PATs places the DHHC-CRD domain in the cytoplasm but such predictions must be confirmed experimentally. B) Each PAT is predicted to span the membrane four to six times; each is ~40 kDa with the greatest degree of sequence variability residing in the cytoplasmic N- and Ctermini. The DHHC-CRD motif defines PATs. Palmitoylation of the cysteine in the DHHC portion is required for transfer of palmitate to a substrate. Most PATs also have a conserved DPG (aspartate-proline-glycine) motif and TTxE (threo‐

In addition to the importance of PAT membrane topology, their membrane system of resi‐ dence is likely to be an important aspect of their function. PATs have been localized to ER, Gol‐ gi, plasma membrane, endosomes, and the yeast vacuole [30, 43-48]; yet, little is known about how these proteins achieve their respective localizations. Immunolocalization of epitope-tag‐ ged DHHC proteins has been somewhat inconsistent among various cell types, between labo‐ ratories (e.g. DHHC2 [30, 37], and even in our own laboratory (unpublished observations SLP) in terms of within which membrane system a protein resides. Such inconsistencies suggest that the cell type, cell cycle, health of the cells, or even the location of the epitope tag may affect sub‐ cellular distribution. An interesting exception is DHHC2. DHHC2 has recently been shown to traffic between the PM and intracellular membranes via recycling endosomes [47]. Important‐ ly, the C-terminal 68 amino acids of DHHC2 was shown to play an important role in defining

nine-threonine-asparagine-glutamate) motif, but their role in the function of PATs is not yet known.

**N-Variable C-Variable**

cytoplasmic tails (Figure 1B).

**<sup>N</sup> <sup>C</sup>**

**DHHC-CRD**

*lumen*

*cytoplasm*

**B.**

**A.**

Hydropathy analyses predict that the PATs encoded by these genes all pass through a mem‐ brane multiple times (at least four) and are expressed predominantly in the ER and Golgi membranes [30, 37, 38]. Currently, there is little published data on the numbers of TM do‐ mains in any of the PATs with the exception of Akr1p in yeast [39]. Predictions using TopPred II 1.1 [40] as presented by Ohno and colleagues [30] show that most PATs have an even number of TM domains with the DHHC-CRD motif in the cytoplasm. However for DHHC13, -16, -11, and -22, the DHHC-CRD motif resides just C-terminal to the first or third TM domain. Assuming the N-terminus is cytoplasmic, this places the DHHC-CRD motif ei‐ ther in the lumen of the ER (the membrane compartment of residence reported for each by Ohno and colleagues 2006) or outside of the cell if expressed on the PM. Given that the envi‐ ronment in these two locations is oxidizing in nature [41, 42] and assuming this topological model is correct, it is possible that the cysteines of the DHHC-CRD motif could form interor intra-molecular disulfide bridges rather than being involved in the transfer of palmitate. However, while it is possible that PATs may assume duties in addition to palmitoylation, it seems somewhat unlikely they would do so in this arrangement as it represents a state in which it would be difficult to perform these functions. The highest-scoring predictions of the membrane topology using TMpred show that the human protein sequence of DHHC11 and -16 should contain four TM domains, DHHC13 eight TM domains, and DHHC22 either four or five TM domains, with each model placing the DHHC-CRD motif inside the cells. There is clear disparity among the predictions generated by the algorithms available and ul‐ timately, any of these predictions of topology must be confirmed or disproved by experi‐ mental data. In any case, for a member of the PAT family to function as a PAT, the DHHC-CRD motif should probably reside in the cytoplasm (Figure 1A). The regions of the PAT proteins that contain the greatest diversity at the amino-acid level are the N- and C-terminal cytoplasmic tails (Figure 1B).

**2.2. The ZDHHC family of PATs**

254 Drug Discovery

The mammalian genome contains at least 23 members of the *ZDHHC* PAT gene family iden‐ tified by the presence of the signature DHHC-cysteine rich domain. Members of the family had been identified as being genes of interest (e.g., "REAM" in metastatic cancer [29]) prior to understanding their function. The genomic structure of *ZDHHC* genes varies widely, in‐ cluding the number and differential use of exons that are spliced together to generate the mRNA. EC gene analyses (http://genome.ewha.ac.kr/ECgene/) of the mRNAs that encode PATs suggest that all of the genes are alternatively spliced at various sites throughout the protein coding sequence as well as within untranslated regions. Many of the putative, alter‐ natively-spliced exons are predicted to encode small peptides that change the structure of the protein in a way that may alter substrate specificity. Similarly, splicing may alter sites for other post-translational modifications, such as phosphorylation or glycosylation, all of which may regulate activity, substrate specificity, subcellular distribution, or interactions with non-substrate proteins. *ZDHHC7*, for example, alters the use of a 111 bp exon that is differentially and specifically expressed in tissues such as placenta, lung, liver, thymus, and small intestine [30]. This exon encodes a 37-residue peptide (EKSSDCRP‐ SACTVKTGLDPTLVGICGEGTESVQSLLL) within the intracellular loop between trans‐ membrane domain 2 (TM2) and TM3 that contains a PKC phosphorylation site. It is conceivable that phosphorylation of this serine changes DHHC7 in such a way that sub‐ strate specificity or the rate of palmitate transfer activity is altered. In addition to alternative mRNA splicing, aberrant splicing induced by mutations or single nucleotide polymor‐ phisms has been shown to occur in at least two ZDHHC genes. A splice-site mutation in highly conserved residues of *ZDHHC9*, a PAT that has been shown to palmitoylate H-Ras and N-Ras [31], has been described in families with X-linked mental retardation (XLMR) [32]. This mutation creates an additional, stronger splice-donor site 140 nt before (toward the 5' end) the normal donor site. Usage of the new site results in a mRNA that is frameshifted and that encodes a truncated protein. Single nucleotide polymorphisms that affect splicing of *ZDHHC8* have also been implicated in schizophrenia [33] (also see: [34-36]).

Hydropathy analyses predict that the PATs encoded by these genes all pass through a mem‐ brane multiple times (at least four) and are expressed predominantly in the ER and Golgi membranes [30, 37, 38]. Currently, there is little published data on the numbers of TM do‐ mains in any of the PATs with the exception of Akr1p in yeast [39]. Predictions using TopPred II 1.1 [40] as presented by Ohno and colleagues [30] show that most PATs have an even number of TM domains with the DHHC-CRD motif in the cytoplasm. However for DHHC13, -16, -11, and -22, the DHHC-CRD motif resides just C-terminal to the first or third TM domain. Assuming the N-terminus is cytoplasmic, this places the DHHC-CRD motif ei‐ ther in the lumen of the ER (the membrane compartment of residence reported for each by Ohno and colleagues 2006) or outside of the cell if expressed on the PM. Given that the envi‐ ronment in these two locations is oxidizing in nature [41, 42] and assuming this topological model is correct, it is possible that the cysteines of the DHHC-CRD motif could form interor intra-molecular disulfide bridges rather than being involved in the transfer of palmitate. However, while it is possible that PATs may assume duties in addition to palmitoylation, it

**Figure 1.** Predicted general structure of PATs. A) The predicted topology of PATs places the DHHC-CRD domain in the cytoplasm but such predictions must be confirmed experimentally. B) Each PAT is predicted to span the membrane four to six times; each is ~40 kDa with the greatest degree of sequence variability residing in the cytoplasmic N- and Ctermini. The DHHC-CRD motif defines PATs. Palmitoylation of the cysteine in the DHHC portion is required for transfer of palmitate to a substrate. Most PATs also have a conserved DPG (aspartate-proline-glycine) motif and TTxE (threo‐ nine-threonine-asparagine-glutamate) motif, but their role in the function of PATs is not yet known.

In addition to the importance of PAT membrane topology, their membrane system of resi‐ dence is likely to be an important aspect of their function. PATs have been localized to ER, Gol‐ gi, plasma membrane, endosomes, and the yeast vacuole [30, 43-48]; yet, little is known about how these proteins achieve their respective localizations. Immunolocalization of epitope-tag‐ ged DHHC proteins has been somewhat inconsistent among various cell types, between labo‐ ratories (e.g. DHHC2 [30, 37], and even in our own laboratory (unpublished observations SLP) in terms of within which membrane system a protein resides. Such inconsistencies suggest that the cell type, cell cycle, health of the cells, or even the location of the epitope tag may affect sub‐ cellular distribution. An interesting exception is DHHC2. DHHC2 has recently been shown to traffic between the PM and intracellular membranes via recycling endosomes [47]. Important‐ ly, the C-terminal 68 amino acids of DHHC2 was shown to play an important role in defining its intracellular localization; however, a defined targeting signal present within this region of DHHC2 and in other DHHC proteins has yet to be defined.

esting to learn if a select subset of cysteines of CD9 is palmitoylated by DHHC2 and also how decreasing palmitoylation of specific cysteines results in the observed cellular behavior. Several other substrates of DHHC2 have been identified ranging from the neuronal adaptor/ scaffold protein PSD95 [62], the SNARE proteins SNAP-23/25 [63], the non-receptor tyrosine kinase Lck [64], and the intracellular signaling proteins Gαi2 [65], GAP43 [62], R7BP [66], and eNOS [48]. Notably, there is no apparent structural similarity between the reported sub‐ strates of DHHC2, or even any sequence similarities surrounding the palmitoylated cysteine residues. Thus, DHHC2 can apparently palmitoylate cysteines located in the N-terminal re‐ gions (PSD-95, GAP-43, and Gα), internally in the protein sequence (SNAP-23/25), in the jux‐ tamembrane region of transmembrane proteins (CD9, CD151, and CKAP4) and close to an

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From these examples, it is clear that upsetting the homeostatic balance of protein palmitoy‐ lation, in either direction, can have significant and deleterious effects on signaling networks. It is also clear that identification of PAT cognate substrates will provide important informa‐ tion concerning the molecular mechanisms underlying the oncogenic nature of the affiliated signaling systems as well as reveal important, novel targets for pharmacologic intervention. The development of specific DHHC protein inhibitors would provide vital reagents with which to study the physiological and pathophysiological importance of many palmitoylated

It is not surprising that a disruption in the homeostatic balance of protein palmitoylation, in either direction, can have pathophysiological consequences. However, one must remain mindful that palmitoylation may not be the sole function of these proteins. Recently, two PATs—HIP14 (DHHC17) and HIP14L (DHHC13)—have been shown to mediate the trans‐ port of Mg2+ [67]. The first indication that these PATs were involved in Mg2+ regulation was that the abundance of their corresponding mRNAs was increased in cells grown in medium with reduced Mg2+ concentration. The authors then showed that Mg2+ (but not Ca2+) trans‐ port was both electrogenic and voltage dependent, and that the transport required palmitoy‐ lation of the PAT. The authors concluded that these two PATs fall into a category of enzymes called "chanzymes" or ion channels that also have enzymatic activity; a type of protein previously represented only by the transient receptor potential melastatin (TRPM) family of transporters [68, 69]. The fact that GODZ (DHHC3) does not appear to mediate Mg2+ transport [70] but can mediate the transport of Ca2+ [71] suggests that this is not a gen‐ eral property of all PATs. The discovery that these PATs transport Mg2+ was astonishing es‐ pecially in light of the fact that the DHHC-CRD motif appears, by sequence and predicted structure, to be a Zn2+-binding protein; (a divalent cation with an atomic radius similar to Mg2+)—not Mg2+. However, Goytain and colleagues also found that HIP14 and HIP14L transported Zn2+ with approximately half the efficiency as Mg2+. The role of these and other PATs in binding to and/or transporting Zn2+ remains to be elucidated, but demonstrates the importance of not limiting ones view of PAT function (or many other proteins for that mat‐

N-terminal myristoylated glycine (Lck and eNOS).

**2.4. PAT functions in addition to palmitoylation**

ter) only to palmitoylation.

proteins and may offer potential for therapeutic development.
