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

its intracellular localization; however, a defined targeting signal present within this region of

PATs have already been linked, in varying degrees, to human disease despite their recent discovery. At least 7 genes encoding PATs have been implicated in human disorders, in‐ cluding *ZDHHC8* with schizophrenia [33], *ZDHHC17* with Huntington's disease [49], *ZDHHC15* and *ZDHHC9* with X-linked mental retardation [32, 50], and *ZDHHC2*, *ZDHHC9*, *ZDHHC17*, and *ZDHHC11* with cancer [29, 51-53]; most of the demonstrated and putative

Overexpression of some PATs has also been shown to alter cancer-related signaling. DHHC17 (HIP14) is oncogenic. DHHC9 and DHHC11 display characteristics that strongly suggest they also are oncogenic. Overexpression of DHHC17 has the ability to induce colo‐ ny formation and anchorage-independent growth in cell culture and tumors in mice [53]. It has been shown that these effects occur, at least in part, by DHHC17-mediated palmitoyla‐ tion of H-, N-, and K2A- RAS proteins [53]. DHHC9 is strongly upregulated in some adeno‐ carcinomas of the gastrointestinal tract at the transcript and protein levels [52] and has also been shown to palmitoylate H- and N- RAS proteins *in vitro* [31]. *ZDHHC11* has a high inci‐ dence of additional genomic copies in cases of non-small cell lung cancer and bladder cancer in which it is strongly linked to high-grade, advanced stage and disease progression [51].

Conversely to the behavior of the oncogenic PATs, a failure to express *ZDHHC2* results in an increase in metastasis in an *in vivo* model leading to the suggestion that *ZDHHC2* is a tu‐ mor/metastasis suppressor [29]. This absence of expression suggests that substrates of DHHC2 are no longer palmitoylated, and that whatever role palmitoylation had in signaling downstream from that event has been disrupted. Such is the case of DHHC2, where due to a lack of palmitoylation, one of its substrates, CKAP4, is no longer normally palmitoylated. One consequence of this is that CKAP4 no longer traffics efficiently (or at all) to the cell sur‐ face where it acts as a receptor for antiproliferative factor (APF) [37] [or presumably its other two known ligands, tissue plasminogen activator [54] and surfactant protein A [55]]. With‐ out surface expression of CKAP4, APF is unable to initiate a wide range of downstream ef‐ fects, including halting cellular proliferation and altering the expression of genes related to

CD9 and CD151, both tetraspanin proteins, have also been identified as substrates of DHHC2 [56]. CD9, which has been suggested to be a tumor suppressor [57, 58], is palmitoy‐ lated on multiple cysteines, but which of these are palmitoylated by DHHC2 is not known. Nonetheless, it is clear that suppression of DHHC2-mediated palmitoylation of CD9 in A431 cells affects cell behaviors that are consistent with it playing a role in tumor suppression. In particular, the cells undergo what appears to be epithelial-mesenchymal transition (EMT) a process in which epithelial cells lose epithelial morphology and markers and gain a fibro‐ blastic morphology during tumor progression [59-61]. It is not yet clear whether this change in cellular behavior was mediated solely by the reduction in CD9 palmitoylation or through reduced palmitoylation of this and other DHHC2 substrates such as CKAP4. It will be inter‐

DHHC2 and in other DHHC proteins has yet to be defined.

**2.3. PAT genes, palmitoylation and human disease**

connections are with cancer.

256 Drug Discovery

the progression of cancer [44].

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‐ ter) only to palmitoylation.

#### **2.5. Enzymatic mechanisms of palmitoylation**

The physical and chemical mechanisms that result in enzymatic palmitoylation have yet to be defined clearly, but some progress has been made using purified proteins. It has been es‐ tablished that mutation of the cysteine in the DHHC motif of all PATs studied to date abol‐ ishes autoacylation of the PAT and palmitoylation of the substrate [23, 56, 62, 72]. This literature as well as discussion of potential physical mechanisms for the reaction have been reviewed recently [3, 73].

with these algorithms is the complexity of the PAT-substrate recognition that is encoded by residues outside of those that immediately surround the palmitoyl-cysteine; the higher or‐

Discovery of Selective and Potent Inhibitors of Palmitoylation

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

259

The unique physical and biochemical nature of the thioester bond that links palmitate to cysteine residues is the basis for the design of many recent assays for palmitoylation. The cysteine residue is among the most nucleophilic entities in a cell [79] and is the most com‐ mon site of palmitoylation. Other residues can be modified by palmitate, but their occur‐ rence is relatively rare and the bond chemistries are different [2, 80-83]. Palmitoylation can also occur in other ways, for example, on an amine of an N-terminal cysteine as is the case with Hedgehog [2, 83, 84], a secreted signaling protein. An example of palmitate modifying the weaker –OH nucleophile of threonine occurs on the carboxyl terminus of a spider toxin [81]. The ε-amino group of lysine can also be modified by palmitate linked by an amide

The reactivity of the thiolate anion of cysteine residues makes it a key component in the structure and function of many proteins by stabilizing higher order structures via disulfide bridges and post-translational modifications like nitrosylation, prenylation, and acylation [85-87]. The high degree of reactivity has also provided a well-characterized, indispensable target for modification by synthetic, thiol-reactive ligands, allowing capture and characteri‐ zation of proteins [88]. An exceptionally useful application of such thiol-specific chemistry is isotope-coded affinity tags (ICAT) for mass spectrometric determination of relative protein or peptide abundance among two or more samples [89-91]. With these probes, changes in abundance of identified proteins or peptides are determined by changes in the ratio of heavy to light-isotope-modified peptides from mixed samples. Combining ICAT technology with functional genomics methods like siRNA-mediated PAT-gene knockdown is one of

In healthy cells the cytoplasm is generally a reducing environment, meaning that solventexposed cysteine side chains are not typically disulfides and thus available to engage in re‐ actions with other molecules [92]. The reactivity of a free cysteine depends on the pKa of the cysteine which is a function of the local environment surrounding the residue within the context of the whole protein. Unlike other residues with nucleophilic side chains (-OH or – NH2), thiol side chains undergo conjugations, redox, and exchange reactions [85]. Conjuga‐ tion reactions (in addition to fatty acylation) include nitric oxide (NO) or S-nitrosylation, re‐ active oxygen species (ROS), and reactive nitrogen species (RNS) forming bonds that are not susceptible to cleavage by hydroxylamine at neutral pH. Hydroxylamine is a reagent used to selectively remove thioester-linked palmitate [93]. Importantly, we know that hydroxyla‐ mine does not perturb disulfides [94], and that it efficiently cleaves thioesters in a quantita‐

In addition to the linkage of palmitate to cysteines, another thioester bond that occurs in cells is the transient association between ubiquitin and the E1, E2, and certain E3 ubiquitina‐ tion enzymes [87, 96]. However, these thioester bonds are easily distinguished from the thio‐

bond. This occurs in several secreted proteins including a bacterial toxin [80].

several mechanisms that will allow us to identify substrates of PATs [37].

der components of the recognition sites.

tive manner [95].

**3.1. The physical properties of cysteines and thioester bonds**
