**4.5. The palmitoyl proteome**

The demonstration that one can effectively replace palmitate with a biotin group led to de‐ velopment of the first, large-scale, proteomic analysis of palmitoylation [74] in yeast, the model system in which the molecular identify of PATs was first determined [23, 26]. This method was dubbed "acyl biotin exchange" or ABE and used the same basic three-steps as described above. As the name implies, the proteins were labeled with a thiol-reactive, bioti‐ nylated heterobifunctional probe, [6-(Biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide (HPDP-biotin), with subsequent capture on streptavidin affinity matrix (for a detailed proto‐ col see Wan et al. 2007 [131]). It is interesting to note the number of proteins that Roth and colleagues captured in the negative control samples (Figure 1a;[74]). The degree of overlap among proteins captured in the experimental and control samples suggests that the step in which free thiols were blocked with NEM was not quantitative and/or that the wash steps following binding of biotinylated proteins to the streptavidin matrix were not sufficiently stringent (steps 7 and 16 respectively from Wan et al, 2007 [131]) thereby resulting in the po‐ tential for a higher number of false-positive hits. However, issues of signal to noise and lim‐ its of sensitivity are by no means unique to this work (avidin-biotin affinity purification is notoriously difficult); rather they are unavoidable issues faced by all developers of novel strategies and users of nascent technologies. Incremental improvements in important assays like this always follow.

live cells or tissue. Next, palmitates are removed from cysteines by cleavage of the thioester bond with hydroxylamine (typically 1.0M) at neutral pH. This creates a new set of free thiols unique in that they were all formerly palmitoylated; ideally, no others should exist. Finally, this new set of formerly-palmitoylated cysteines is modified by any one of the many thiolspecific reagents. The uniqueness of the individual assays that incorporate these steps lies primarily in the choice of thiol-specific reagents, and this choice depends on what questions the investigator wants to answer. There are also variations in the reagents used to block free cysteines in the first step. Both NEM and MMTS have been used in the assays described be‐

Cysteines that are palmitoylated can also be modified by fatty acids other that palmitate [7] including stearate and oleate. The acyl-exhange method cannot yet distinguish between pal‐ mitate and the other fatty acids modifying cysteines by a thioester bond. Two additional points that relate to the specificity of this method for palmitoylation are: 1) that it will not report modification of cysteines by prenyl groups (geranylgeranyl or farnesyl) because they are attached by a thioether bond that is not susceptible to cleavage by hydroxylamine and 2) it will not report myristoylated proteins because this 14-carbon acyl group is linked to an Nterminal glutamate by an amide bond which is also insensitive to cleavage by hydroxyla‐

The recent development of novel assays using the three-step acyl exchange method to study palmitoylation in a broader sense was invigorated by two publications describing a new twist on the method that incorporated the use of radiolabeled NEM assay [129, 130]. Work described in these papers showed that labeling palmitoyl cysteines with radiolabeled NEM resulted in a remarkable 5- to 12-fold increase in sensitivity to detect several known palmito‐

In addition, the authors demonstrated the utility of the biotinylated, heterobifunctional crosslinker, 4-[4′-(maleimidomethyl)cyclohexanecarboxamido] butane (Btn-BMCC), as an ef‐ fective tool to capture and purify (using streptavidin-agarose) palmitoylated proteins. In do‐ ing so, they also demonstrated the general potential of using the wide variety of existing thiol-specific probes for the development of additional assays for palmitoylation that are be‐

The demonstration that one can effectively replace palmitate with a biotin group led to de‐ velopment of the first, large-scale, proteomic analysis of palmitoylation [74] in yeast, the model system in which the molecular identify of PATs was first determined [23, 26]. This method was dubbed "acyl biotin exchange" or ABE and used the same basic three-steps as described above. As the name implies, the proteins were labeled with a thiol-reactive, bioti‐ nylated heterobifunctional probe, [6-(Biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide (HPDP-biotin), with subsequent capture on streptavidin affinity matrix (for a detailed proto‐ col see Wan et al. 2007 [131]). It is interesting to note the number of proteins that Roth and colleagues captured in the negative control samples (Figure 1a;[74]). The degree of overlap among proteins captured in the experimental and control samples suggests that the step in

H-palmitate.

yl proteins, including PSD-95 and SNAP-25, when compared to labeling with 3

low but NEM is used most commonly.

mine.

264 Drug Discovery

ginning to materialize.

**4.5. The palmitoyl proteome**

One of the key features of all proteomic methods is the system used for detection of specifi‐ cally-isolated proteins or peptides. Work by Roth et al. [74] identified proteins by multi-di‐ mensional protein identification technology (MudPIT), a high-throughput, tandem mass spectrometry (MS/MS)-based proteomic technology [132] [see also [131, 133]]. Compared to other mass spectrometric methods, MudPIT has the potential to identify less abundant pro‐ teins with a higher degree of confidence, because multiple peptides of a single protein can be used to identify a protein of interest. One downside with MudPIT in this case is that the palmitoyl cysteine(s) cannot be pinpointed, as there may be many candidates among the in‐ dividual peptides of a whole protein suspected as being a palmitoyl protein. After demon‐ strating the usefulness of this large-scale method for purification and identification of palmitoylated proteins, the authors used mutant strains of yeast lacking one or more of the seven yeast PAT proteins to identify substrates of individual PATs. Comparison of the de‐ gree of palmitoylation of individual proteins between wild type yeast (a full set of normally palmitoylated proteins) and those not expressing one or more of the yeast PATs (each with a specific set of hypo/depalmitoylated proteins) provided the identity of the substrates of indi‐ vidual PATs. Together, this work represents a very significant contribution to the identifica‐ tion and understanding of the yeast palmitoyl proteome and provided many important clues about potential homologous PAT-substrate pairs in other systems.

The complexity of palmitoylation is greater in a vertebrate system. With at least 23 genes en‐ coding PATs identified in humans, the diversity at the most basic level is at least three-fold greater than in yeast. When one considers the additional variants encoded by alternative splicing of PAT mRNAs, the potential diversity increases even more. The greater number of PATs suggests (but does not prove) that there are also more palmitoylated proteins in mam‐ mals. The ability to genetically manipulate mammalian cells is improving but lags behind yeast. Nevertheless, defining the palmitoyl proteome or palmitoylosome and how it is regu‐ lated in mammals (humans in particular) is a task of significant importance and interest. Now that the enzymes capable of mediating palmitoylation have been identified, one of the most important questions that we face is which substrates are palmitoylated by each PAT a question brought sharply into focus when one considers the known connections between mutations or deletions in PAT genes and human disease, in particular cancer. DHHC2 is de‐ leted in many types of cancer (see above). Its absence is strongly correlated with an increase in the metastatic potential of cancer cells. The simplest inverse corollary in this case is that palmitoylated substrates of DHHC2 are responsible for keeping cells from metastasizing. Identification of these substrates and their associated signaling networks using novel assays for palmitoylation has begun to provide supporting evidence for known mechanisms of can‐ cer progression [56] as well as a novel signaling pathway for the regulation of cellular prolif‐ eration and metastasis [37].

vides a mechanism for affinity purification of thiol-captured peptides on an avidin column. (D) Proteins from knockdown and control conditions are mixed in equal amounts and digested in-gel with trypsin. ICAT labeled pepti‐ des are enriched by avidin affinity and analyzed by LC/MS. A pair of ICAT-labeled peptides is chemically identical and is easily visualized, because they essentially coelute and there is a 9 Da mass difference measured in a scanning mass spectrometer. Even if equal amounts of a single protein exist in two different samples, the quantity of protein that is captured depends directly on its degree of palmitoylation; if all of a single protein is palmitoylated under one condi‐ tion, then all of it will be captured; if only half of this protein is palmitoylated under another condition then the cap‐ ture rate of that protein will be half as much, relative to control, making it appear half as abundant. Proteins for which there has been no change in palmitoylation (ie, equal capture rates) will yield a heavy:light (H/L) ratio of 1. The degree to which palmitoylation is diminished will register as a decrease in the H/L ratio (ie, 50% reduction in palmitoylation will correspond to a H/L ratio of 0.5). A change in the capture rate that results in a change in the post-purification abundance is measured in the LC/MS phase. (E) Finally, the peptides are further fragmented into their constituent

Discovery of Selective and Potent Inhibitors of Palmitoylation

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267

amino acids by MS/MS, enabling identification of the proteins corresponding to the captured peptides.

**substrates and palmitoyl proteins in vertebrates**

tures that will be discussed below.

**4.6. Palmitoyl-cysteine Identification Capture and Analysis (PICA): Identification of PAT**

With the aim of defining PAT-substrate specificity in a living vertebrate system, we devel‐ oped a method to identify substrates of specific PATs in mammalian cells and tissues called Palmitoyl cysteine Isolation Capture and Analysis or PICA [37]. We used this method to identify CKAP4/p63, a known palmitoyl protein [134] as one substrate of DHHC2 in HeLa cells [37]. This method is similar to ABE as described by Roth et al (2006) but was inspired [135] in part by the work of Drisdel and Green (2004) and incorporated several novel fea‐

The ability of PICA to identify PAT substrates is based on the principle that it quantifies the differential frequency of palmitoylation of individual proteins or peptides in control condi‐ tions versus conditions in which the function of a single PAT is reduced by siRNA-mediated gene knockdown. The process to identify substrates of DHHC2 consisted of four basic steps outlined in Figure 2. In the first part we generated two distinct pools of palmitoylated pro‐ teins, one from control HeLa cells and the other from HeLa cells in which the activity of one PAT (DHHC2) was reduced. These two distinct pools of palmitoylated proteins were then captured and compared directly to identify differences in the degree of palmitoylation of in‐ dividual proteins between the two pools. To do this, we reduced the expression of *ZDDHC2* mRNA (and consequently the abundance of the encoded enzyme, DHHC2) in HeLa cells us‐ ing siRNA-mediated gene knockdown which resulted in a reduced level of palmitoylation of DHHC2 substrates. Total protein from knockdown and control cells was prepared by first blocking free thiols with MMTS in the presence of SDS. This was followed by selective expo‐ sure of all palmitoyl cysteines by cleavage of the palmitoyl-cysteine thioester bond with 1.0M hydroxylamine at neutral pH, thereby generating a unique population of formerly pal‐ mitoylated cysteines. Second, we selectively and differentially labeled the exposed, former‐ ly-palmitoylated cysteines from knockdown and control cells with biotinylated, thiolreactive heavy (H) and light (L) ICAT reagents, respectively. Third, we combined equal quantities of the ICAT-labeled protein from *ZDHHC2* knockdown and control cells and di‐ gested the mixture with trypsin. The resulting H and L ICAT-labeled tryptic peptides were captured and purified on an avidin affinity column. Finally, ICAT-labeled, putative, former‐ ly-palmitoylated peptides were analyzed by mass spectrometry. Peptides with a reduced

**Figure 2.** Palmitoyl-cysteine Identification Capture and Analysis (PICA): Determining PAT-substrate specificity by dif‐ ferential labeling of palmitoylated proteins with Isotope-coded Affinity Tags (ICAT) (A) In one set of cultures, *ZDHHC2* expression is knocked down by transfecting HeLa cells with *ZDHHC2*-specific siRNA (Dharmacon). Proteins are extract‐ ed from experimental and control cells and treated with the thiol-specific blocking reagent MMTS. This step chemical‐ ly modifies or protects all thiols ("X" on the proteins P1-P3) that are free at physiological pH and leaves the palmitoylated cysteines (P) undisturbed as depicted on P1-P3. (B) Following the protection or blocking of free thiols, palmitates are removed by selective cleavage of the thioester bond with hydroxylamine at pH 7.4, which generates a distinctive set of free, formerly-palmitoylated, reactive thiols (C) that can be selectively labeled with ICAT reagents. Io‐ doacetamide at one end of the ICAT reagent binds to the thiol sidechain of cysteines; on the other end, biotin pro‐

vides a mechanism for affinity purification of thiol-captured peptides on an avidin column. (D) Proteins from knockdown and control conditions are mixed in equal amounts and digested in-gel with trypsin. ICAT labeled pepti‐ des are enriched by avidin affinity and analyzed by LC/MS. A pair of ICAT-labeled peptides is chemically identical and is easily visualized, because they essentially coelute and there is a 9 Da mass difference measured in a scanning mass spectrometer. Even if equal amounts of a single protein exist in two different samples, the quantity of protein that is captured depends directly on its degree of palmitoylation; if all of a single protein is palmitoylated under one condi‐ tion, then all of it will be captured; if only half of this protein is palmitoylated under another condition then the cap‐ ture rate of that protein will be half as much, relative to control, making it appear half as abundant. Proteins for which there has been no change in palmitoylation (ie, equal capture rates) will yield a heavy:light (H/L) ratio of 1. The degree to which palmitoylation is diminished will register as a decrease in the H/L ratio (ie, 50% reduction in palmitoylation will correspond to a H/L ratio of 0.5). A change in the capture rate that results in a change in the post-purification abundance is measured in the LC/MS phase. (E) Finally, the peptides are further fragmented into their constituent amino acids by MS/MS, enabling identification of the proteins corresponding to the captured peptides.

for palmitoylation has begun to provide supporting evidence for known mechanisms of can‐ cer progression [56] as well as a novel signaling pathway for the regulation of cellular prolif‐

**Figure 2.** Palmitoyl-cysteine Identification Capture and Analysis (PICA): Determining PAT-substrate specificity by dif‐ ferential labeling of palmitoylated proteins with Isotope-coded Affinity Tags (ICAT) (A) In one set of cultures, *ZDHHC2* expression is knocked down by transfecting HeLa cells with *ZDHHC2*-specific siRNA (Dharmacon). Proteins are extract‐ ed from experimental and control cells and treated with the thiol-specific blocking reagent MMTS. This step chemical‐ ly modifies or protects all thiols ("X" on the proteins P1-P3) that are free at physiological pH and leaves the palmitoylated cysteines (P) undisturbed as depicted on P1-P3. (B) Following the protection or blocking of free thiols, palmitates are removed by selective cleavage of the thioester bond with hydroxylamine at pH 7.4, which generates a distinctive set of free, formerly-palmitoylated, reactive thiols (C) that can be selectively labeled with ICAT reagents. Io‐ doacetamide at one end of the ICAT reagent binds to the thiol sidechain of cysteines; on the other end, biotin pro‐

eration and metastasis [37].

266 Drug Discovery

#### **4.6. Palmitoyl-cysteine Identification Capture and Analysis (PICA): Identification of PAT substrates and palmitoyl proteins in vertebrates**

With the aim of defining PAT-substrate specificity in a living vertebrate system, we devel‐ oped a method to identify substrates of specific PATs in mammalian cells and tissues called Palmitoyl cysteine Isolation Capture and Analysis or PICA [37]. We used this method to identify CKAP4/p63, a known palmitoyl protein [134] as one substrate of DHHC2 in HeLa cells [37]. This method is similar to ABE as described by Roth et al (2006) but was inspired [135] in part by the work of Drisdel and Green (2004) and incorporated several novel fea‐ tures that will be discussed below.

The ability of PICA to identify PAT substrates is based on the principle that it quantifies the differential frequency of palmitoylation of individual proteins or peptides in control condi‐ tions versus conditions in which the function of a single PAT is reduced by siRNA-mediated gene knockdown. The process to identify substrates of DHHC2 consisted of four basic steps outlined in Figure 2. In the first part we generated two distinct pools of palmitoylated pro‐ teins, one from control HeLa cells and the other from HeLa cells in which the activity of one PAT (DHHC2) was reduced. These two distinct pools of palmitoylated proteins were then captured and compared directly to identify differences in the degree of palmitoylation of in‐ dividual proteins between the two pools. To do this, we reduced the expression of *ZDDHC2* mRNA (and consequently the abundance of the encoded enzyme, DHHC2) in HeLa cells us‐ ing siRNA-mediated gene knockdown which resulted in a reduced level of palmitoylation of DHHC2 substrates. Total protein from knockdown and control cells was prepared by first blocking free thiols with MMTS in the presence of SDS. This was followed by selective expo‐ sure of all palmitoyl cysteines by cleavage of the palmitoyl-cysteine thioester bond with 1.0M hydroxylamine at neutral pH, thereby generating a unique population of formerly pal‐ mitoylated cysteines. Second, we selectively and differentially labeled the exposed, former‐ ly-palmitoylated cysteines from knockdown and control cells with biotinylated, thiolreactive heavy (H) and light (L) ICAT reagents, respectively. Third, we combined equal quantities of the ICAT-labeled protein from *ZDHHC2* knockdown and control cells and di‐ gested the mixture with trypsin. The resulting H and L ICAT-labeled tryptic peptides were captured and purified on an avidin affinity column. Finally, ICAT-labeled, putative, former‐ ly-palmitoylated peptides were analyzed by mass spectrometry. Peptides with a reduced H/L ratio over four independent runs were analyzed further to confirm that the identified cysteine was indeed palmitoylated by DHHC2 under physiological conditions. Details of the protocol and reagents used and outlined in Figure 2 can be found in [37].

co-overexpression of one PAT and the substrate (for a review see: [72]). Using this method, an increase in the incorporation of radiolabled palmitate on the overexpressed substrate in the presence of an overexpressed PAT is used to claim specificity. This method is an impor‐ tant tool for increasing our understanding of palmitoylation-related phenomena including confirmation of putative PAT-substrate pairs identified by other methods. Likewise, when starting with a known palmitoyl protein and the intention of identifying the PAT responsi‐ ble for its palmitoylation, it remains a useful method. However, we should remember that just because overexpression of a PAT can increase the incorporation of palmitate onto a spe‐ cific protein does not necessarily mean that it does so in a live cell. Again, problems like this are not unique to this method and simply reflect our lack of knowledge about where and when PATs and their substrates are expressed, the degree of promiscuity among PATs and,

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269

The potential for specific cysteines to be modified by both palmitate and RA via a thioester bond is an issue that deserves attention from those of us interested primarily in palmitoyla‐ tion for at least two reasons. One is the potential that an exchange between the two modifi‐ cations is a physiologically relevant means of regulating signaling and second, the possibility that proteins identified as being palmitoylated in assays utilizing some form of

This particularly interesting approach labels cysteines with isosteric, azido-derivitives of fatty acids that are able to substitute for fatty acids that occur naturally in cells (Figure 3) [112, 136]. Once bound to cysteines, the azido group on the fatty acid is reacted, with a high degree of selectivity, via the Staudinger reaction [137] with (triaryl)phosphines that are themselves derivatized with Myc, biotin, a fluorophore, or others. Using this meth‐ od Hang and colleagues [112] found that ω-azido fatty acids with 12 and 15 carbons can be efficiently metabolized by mammalian cells and accurately report myristoylation and

Work by Kostiuk and colleagues identified palmitoyl proteins from mitochondria us‐ ing azido-palmitate [136]. To accomplish this they purified proteins from cellular mito‐ chondrial fractions, first by differential centrifugation, then by further purification based first on charge and subsequently by size using chromatographic separation. Labeling of proteins in this study was outside of a living system presumably leaving only the pos‐ sibility of autocatalytic/non-enzymatic palmitoylation. Mass spectrometric analysis of se‐ lected bands identified 21 palmitoylated proteins, 19 of which were novel. The majority of the proteins labeled were metabolic-type proteins unique to the mitochondrion. This raises the interesting possibility that the principle mechanism of palmitoylation in this organelle is autocatalytic rather than enzymatic, and that, as suggested by the au‐ thors, the key role of palmitoylation in the mitochondrion is to inhibit enzymes by pal‐

These so called bio-orthogonal probes have been reported to be nontoxic and very stable un‐ der physiological conditions. Importantly, this two-step reaction is rapid and more sensitive

how PAT function is regulated.

ABE chemistry are RA-modified instead.

thio-palmitoylation, respectively.

**4.8. Labeling palmitoyl proteins with bioorthogonal probes**

mitoylation of cysteines in the vicinity of the active site.

There are several unique aspects in the PICA method. First, we used MMTS to block the free thiols in the first step. NEM is used most commonly at this step, but MMTS is more reactive and smaller than NEM or iodoacetamide, enhancing its ability to modify all free reactive cysteines. Inefficient blocking of free thiols in the first step is one factor that could easily contribute to false-positive capture of proteins in the purification step. Qualitative evalua‐ tion (silver-stained SDS-PAGE) of protein capture in experimental and control (no-hydrox‐ ylamine) conditions [Figure 2, [37]] suggests that it may be more efficient than NEM (for comparison see Figure 1A [74]). However, it may also be that we captured very few proteins under control conditions because of a more stringent wash protocol than described by Roth et al [74]. The use of ICAT reagents in PICA allowed us to combine formerly-palmitoylated peptides purified from control and experimental cells in the same pool, and subsequently, a direct, simultaneous analysis of palmitoylation in the two pools in a single analytical sam‐ ple. We defined a substrate of DHHC2 as one that had a consistently reduced H/L ratio over four independent PICA runs. This approach provided us with many (the vast majority), con‐ venient internal control peptides which are peptides that were not substrates of DHHC2 that had unchanged H/L ratios. This approach significantly reduces the potential for identi‐ fication of false-positive hits because, if a protein can be falsely labeled by an ICAT, it should do so with equal efficiency in both the control and experimental cells yielding a pep‐ tide with an H/L ratio of ~1. The greater risk with this approach is the failure to identify sub‐ strates that exist in low abundance. Using tandem mass spectrometry, we analyzed a sample of significantly reduced complexity including only ICAT-tagged peptides. As is inherent in such analyses, the most abundant peptides dominate the report. However, one advantage of this approach is that when a peptide is identified, whether it is a substrate of a single PAT or not, the palmitoyl cysteine(s) is also identified. In the case of CKAP4/p63 (and the majority of other peptides) there was only a single cysteine, and it was already known to be a site for palmitoylation [134]. Spectral counting has the potential to positively identify palmitoyl pro‐ teins of lower abundance because more than a single peptide from any given protein is fac‐ tored into to the identification. There is greater overall coverage (identified peptide fragments of a protein) using this method thereby increasing the confidence level of identifi‐ cation. However, the disadvantage inherent in analyzing a complex mixture, including nonpalmitoylated peptides by spectral counting, is that identification of the palmitoyl cysteine (in the cases where there are multiple candidate cysteines) must await subsequent and tedi‐ ous analyses. The tradeoff between these two complementary approaches in mass spectro‐ metric analysis is sensitivity versus specificity. Combining these analyses will provide a much greater depth of coverage.

#### **4.7. Forward and reverse approaches to assigning PAT-substrate pairs**

The first reports that identified PAT-substrate pairings took the reverse approach: start with a known palmitoylated protein then use metabolic labeling with radiolabeled palmitate and co-overexpression of one PAT and the substrate (for a review see: [72]). Using this method, an increase in the incorporation of radiolabled palmitate on the overexpressed substrate in the presence of an overexpressed PAT is used to claim specificity. This method is an impor‐ tant tool for increasing our understanding of palmitoylation-related phenomena including confirmation of putative PAT-substrate pairs identified by other methods. Likewise, when starting with a known palmitoyl protein and the intention of identifying the PAT responsi‐ ble for its palmitoylation, it remains a useful method. However, we should remember that just because overexpression of a PAT can increase the incorporation of palmitate onto a spe‐ cific protein does not necessarily mean that it does so in a live cell. Again, problems like this are not unique to this method and simply reflect our lack of knowledge about where and when PATs and their substrates are expressed, the degree of promiscuity among PATs and, how PAT function is regulated.

The potential for specific cysteines to be modified by both palmitate and RA via a thioester bond is an issue that deserves attention from those of us interested primarily in palmitoyla‐ tion for at least two reasons. One is the potential that an exchange between the two modifi‐ cations is a physiologically relevant means of regulating signaling and second, the possibility that proteins identified as being palmitoylated in assays utilizing some form of ABE chemistry are RA-modified instead.

### **4.8. Labeling palmitoyl proteins with bioorthogonal probes**

H/L ratio over four independent runs were analyzed further to confirm that the identified cysteine was indeed palmitoylated by DHHC2 under physiological conditions. Details of the

There are several unique aspects in the PICA method. First, we used MMTS to block the free thiols in the first step. NEM is used most commonly at this step, but MMTS is more reactive and smaller than NEM or iodoacetamide, enhancing its ability to modify all free reactive cysteines. Inefficient blocking of free thiols in the first step is one factor that could easily contribute to false-positive capture of proteins in the purification step. Qualitative evalua‐ tion (silver-stained SDS-PAGE) of protein capture in experimental and control (no-hydrox‐ ylamine) conditions [Figure 2, [37]] suggests that it may be more efficient than NEM (for comparison see Figure 1A [74]). However, it may also be that we captured very few proteins under control conditions because of a more stringent wash protocol than described by Roth et al [74]. The use of ICAT reagents in PICA allowed us to combine formerly-palmitoylated peptides purified from control and experimental cells in the same pool, and subsequently, a direct, simultaneous analysis of palmitoylation in the two pools in a single analytical sam‐ ple. We defined a substrate of DHHC2 as one that had a consistently reduced H/L ratio over four independent PICA runs. This approach provided us with many (the vast majority), con‐ venient internal control peptides which are peptides that were not substrates of DHHC2 that had unchanged H/L ratios. This approach significantly reduces the potential for identi‐ fication of false-positive hits because, if a protein can be falsely labeled by an ICAT, it should do so with equal efficiency in both the control and experimental cells yielding a pep‐ tide with an H/L ratio of ~1. The greater risk with this approach is the failure to identify sub‐ strates that exist in low abundance. Using tandem mass spectrometry, we analyzed a sample of significantly reduced complexity including only ICAT-tagged peptides. As is inherent in such analyses, the most abundant peptides dominate the report. However, one advantage of this approach is that when a peptide is identified, whether it is a substrate of a single PAT or not, the palmitoyl cysteine(s) is also identified. In the case of CKAP4/p63 (and the majority of other peptides) there was only a single cysteine, and it was already known to be a site for palmitoylation [134]. Spectral counting has the potential to positively identify palmitoyl pro‐ teins of lower abundance because more than a single peptide from any given protein is fac‐ tored into to the identification. There is greater overall coverage (identified peptide fragments of a protein) using this method thereby increasing the confidence level of identifi‐ cation. However, the disadvantage inherent in analyzing a complex mixture, including nonpalmitoylated peptides by spectral counting, is that identification of the palmitoyl cysteine (in the cases where there are multiple candidate cysteines) must await subsequent and tedi‐ ous analyses. The tradeoff between these two complementary approaches in mass spectro‐ metric analysis is sensitivity versus specificity. Combining these analyses will provide a

protocol and reagents used and outlined in Figure 2 can be found in [37].

268 Drug Discovery

much greater depth of coverage.

**4.7. Forward and reverse approaches to assigning PAT-substrate pairs**

The first reports that identified PAT-substrate pairings took the reverse approach: start with a known palmitoylated protein then use metabolic labeling with radiolabeled palmitate and

This particularly interesting approach labels cysteines with isosteric, azido-derivitives of fatty acids that are able to substitute for fatty acids that occur naturally in cells (Figure 3) [112, 136]. Once bound to cysteines, the azido group on the fatty acid is reacted, with a high degree of selectivity, via the Staudinger reaction [137] with (triaryl)phosphines that are themselves derivatized with Myc, biotin, a fluorophore, or others. Using this meth‐ od Hang and colleagues [112] found that ω-azido fatty acids with 12 and 15 carbons can be efficiently metabolized by mammalian cells and accurately report myristoylation and thio-palmitoylation, respectively.

Work by Kostiuk and colleagues identified palmitoyl proteins from mitochondria us‐ ing azido-palmitate [136]. To accomplish this they purified proteins from cellular mito‐ chondrial fractions, first by differential centrifugation, then by further purification based first on charge and subsequently by size using chromatographic separation. Labeling of proteins in this study was outside of a living system presumably leaving only the pos‐ sibility of autocatalytic/non-enzymatic palmitoylation. Mass spectrometric analysis of se‐ lected bands identified 21 palmitoylated proteins, 19 of which were novel. The majority of the proteins labeled were metabolic-type proteins unique to the mitochondrion. This raises the interesting possibility that the principle mechanism of palmitoylation in this organelle is autocatalytic rather than enzymatic, and that, as suggested by the au‐ thors, the key role of palmitoylation in the mitochondrion is to inhibit enzymes by pal‐ mitoylation of cysteines in the vicinity of the active site.

These so called bio-orthogonal probes have been reported to be nontoxic and very stable un‐ der physiological conditions. Importantly, this two-step reaction is rapid and more sensitive than labeling with 125I-palmitoyl-CoA. These features, especially their ability to effectively substitute for endogenous fatty acids, make them ideal for labeling palmitoyl proteins in live cells, providing a significantly more direct measure of protein palmitoylation than can be achieved in any other assay format. It is easy to imagine that use of such probes will come to dominate in experimental systems for studying palmitoylation.

and causes death even after a brief exposure to 100 µM. 2BP inhibits several enzymes in‐ volved in lipid metabolism, including carnitine palmitoyltransferase 1, fatty acid CoA ligase, glycerol-3-phosphate acyltransferase, and enzymes in the synthesis of triacylglycerol bio‐ synthesis [140, 141]. This high degree of promiscuity as well as the toxicity of 2BP renders it nearly useless as a tool to determine anything specific about palmitoylation related signal‐ ing issues that equally plague cerulenin and tunicamycin. The uses and effects of these three

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Smith and colleagues [126] recently screened a compound library in an attempt to identify more selective and potent inhibitors of palmitoylation, in particular inhibitors of PATs. This screen identified single compounds from five chemical classes (Compounds I-V) that inhib‐ ited cellular processes associated with palmitoylation. The assays used in the screens includ‐ ed: measuring the *in vivo* and *in vitro* growth rate of an NIH/3T3 cell line that overexpressed DHHC17, displacement from the plasma membrane of myristoylated or farnesylated GFP, and *in vitro* palmitoylation of small, non-complex, myristoylated or farnesylated, synthetic, fluorescent peptides intended to mimic known palmitoylation substrates [123, 126, 127]. These assays could not discriminate a direct effect of any compound on any PAT. They could only report the activity of compounds that acted at some point (not excluding direct PAT inhibition) in any pathway that leads to or affects palmitoylation; compounds like 2BP, cerulenin, and tunicamycin. This assertion was borne out in follow-up studies on the same compounds [138] (see below). Perhaps the most intriguing finding in this report was that compounds I-IV were able to suppress the oncogenic behavior of human cells that overex‐

inhibitors was reviewed recently [142].

2-bromopalmitate

tunicamycin

cerulenin

**Figure 4.** Lipid-based inhibitors of palmitoylation.

**Figure 3.** Using Click chemistry and bio-orthogonal probes to label palmitoyl cysteines. A) A palmitoylated protein; the shaded box indicates the thioester bond. B) Azido-palmitate is transferred to a protein forming a thioester bond with a cysteine residue. The azide moiety of the azido-palmitate reacts, via the Staudinger reaction, with the tagged (in this case biotin) phosphine, forming an amide bond. The biotin-tagged proteins can then be affinity purified and analyzed in various ways including mass spectrometry. Tags and reporters other than biotin can be added to the phosphine providing a wide array of potential methods for subsequent analyses.
