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

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 connections are with cancer.

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 the progression of cancer [44].

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‐ 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 N-terminal myristoylated glycine (Lck and eNOS).

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 proteins and may offer potential for therapeutic development.
