**4. Novel methods to discover and identify PAT/substrate specificity**

The chemistry supporting novel assays to study palmitoylation and the reagents that are be‐ ing incorporated into them have, for the most part, been known and available for years [88] . Most of the methods that are now being developed to study palmitoylation capitalize on many years of knowledge and development of cysteine-specific chemistries, developed mainly as methods to purify and/or specifically target proteins and peptides with various reagents. Many of the reagents that specifically label cysteines have been created as both af‐ finity and fluorescent tags, the former for purification and structure determinations [88]and the latter as cellular reporters of protein abundance, subcellular distribution, protein confor‐ mation changes, the formation of the Golgi, and even the concentration of cellular analytes in specific subcellular domains. The following references provide a short list of some of the most clever uses of thiol chemistry [113-119]. Given the wealth of information on the unique chemistry of the palmitoyl thioester bond and the tools for capturing and characterizing cys‐ teines in proteins, it is somewhat surprising that we are only now developing innovative as‐ says to increase our understanding of palmitoylation. This recent increase is most likely tied to the dramatic increase in the utility of mass spectrometry as a proteomic tool. To provide a general frame of reference for the recent shift in the types of assays that are being devel‐ oped, we will briefly discuss other assays that have been used successfully for a longer peri‐ od of time. These assays are by no means outdated and some continue to be the most appropriate way to answer specific questions.

versed in 0.1N HCl and reducing reagents like dithiothreitol (DTT) but apparently not by TCEP. Phenylmercury derivatives also react faster with thiols than do the commonly used

Compounds containing disulfide bonds are able to undergo disulfide exchange reactions with another thiol by the free thiol attacking the disulfide bond and subsequent formation of a new mixed thiol. Two examples of useful compounds in this category are Methylmethane‐ thiosulfonate (MMTS) and pyridyl disulfide derivitives like biotin HPDP ((*N*-(6-(Biotinami‐ do)hexyl)-3'-(2'-pyridyldithio)-propionamide). MMTS can be used in some cases to block free thiols more effectively than NEM, as it is uncharged and thus more likely to modify all free reactive cysteines. MMTS has been shown not to react with nitrosothiols or existing di‐

The most common method to identify palmitoyl proteins and to determine the residence half-life of palmitate on a specific protein or palmitate turnover (e.g. [122] for a particularly interesting example) is to metabolically label cells with radiolabled palmitate. 14C-, 3

tively inexpensive and widely available. However, using 125I -labeled palmitate provides some advantages. In practical terms, the time required for detection is considerably shorter -

patible with phosphorimaging technology which is much more rapid and quantitative than densitometric measurements from films generated by autofluorography (as is used with tri‐ tium). The principle downside of using 125I-labeled palmitate is that it is not commercially available, and the labeling must be done by the investigator. Reviews of the methods using radiolabeled palmitate and including technical details have been published recently

Fluorescently-labeled peptides that mimic PAT substrates have been used to characterize PAT activity and for the discovery of inhibitors of palmitoylation [123, 126, 127]. The use of these peptides over the last several years was reviewed recently [123]. Peptide substrates for palmitoylation have also been genetically fused to fluorescent proteins and expressed in cells. This strategy has been used to determine how palmitoylation affects subcellular traf‐ ficking both between and within membranes [124]. Monomeric GFP-based reporters and fluorescence resonance energy transfer proved to be helpful in the identification of lipid

Most of the novel assays for palmitoylation utilize the same basic foundation first described for a palmitoyl protein by Schmidt and colleagues [128] and now most commonly known as acyl-exchange. First, free cysteines are blocked on proteins that have been extracted from

rafts with an affinity for palmitate on the inner leaflet of the plasma membrane [5].

H- and

H-palmitate is most common because it is rela‐

Discovery of Selective and Potent Inhibitors of Palmitoylation

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

263

H-palmitate. The *γ-*irradiation from the 125I is also com‐

thiol-reactive *N*-ethylmaleimide (NEM).

**4.2. Metabolic labeling with radiolabeled palmitate**

**4.3. Fluorescently-labeled peptide substrates for palmitoylation**

125I-labeled palmitate have all been used, but 3

hours instead of (often) weeks with 3

**4.4. Acyl-biotin exchange: ABE**

sulfides [121].

[123-125].

#### **4.1. Chemistry and physical properties of palmitoyl cysteines: Reactions and probes**

Working with palmitoylated proteins is inherently difficult due to the labile nature of the thioester bond and the increased hydrophobicity of the protein or peptide due to palmitate. On the other hand, the unique physical and chemical properties of thiols, palmitoylated thi‐ ols, and the thioester bond make them particularly amenable to modification by highly spe‐ cific chemistry and a wide variety of thiol reactive probes.

#### *Reactions of free thiols in the cytoplasm*

Thiol modification occurs most commonly in cells by one of two routes: disulfide exchange or alkylation. Many of the reactive groups that undergo these two reactions are relatively stable in aqueous environments; the reactions are rapid and provide high yields of thioether and disulfide bonds [88]. Thiols will also react with many amine reactive reagents including isothiocyanates and succinimidyl esters but lack a high degree of specificity, resulting in un‐ stable bonds that are much less useful for routine modification of thiols in proteins. Thiolspecific reagents and chemistry figure strongly into the design and development of novel assays for palmitoylation. Most investigators are limited somewhat to reagents that are available from a catalog but, fortunately, there are already many useful reagents available. Among the most useful are thio-reactive chemicals that are linked to another moiety (reac‐ tive or reporter) by a spacer arm of variable length and physical characteristics. Such heteroand homo-bifunctional crosslinking reagents have provided much of the foundation for recent developments in palmitoylation assays and provide a fairly rich toolbox for future as‐ say development.

#### *Chemical moieties that react with palmitoyl-cysteines*

Iodoacetamide conjugates are among the most commonly used tools for modifying cysteine thiols. These undergo nucleophilic substitution to form stable thioether bonds at physiologi‐ cal pH in aqueous environments. When using iodoacetamide and its conjugates, one should remember that depending on the pH of the solution, they can also react with histidine, ly‐ sine, and methionine (at pH >1.7) residues and N-terminal amines. However, when used at slightly alkaline pH in the dark and in the absence of reducing reagents, cysteine modifica‐ tion will be the exclusive reaction [88]. A good example of iodoacetamide-based probes are the isotope-coded affinity tags or ICAT [120]. These have proved particularly useful in de‐ termining the substrates of DHHC2 [37].

Maleimides are also common constituents of heterobifunctional crosslinking reagents and blocking reagents that target cysteines. They are ~1000 times more specific for cysteine sulf‐ hydryls at pH 6.5-7.5, but at higher pH some cross reactivity can occur with amines. Mal‐ eimides form stable thioether bonds by adding the sulfhydryl across the double bond of the maleimide.

Phenylmercury derivatives react with thiols, including nitrosothiols, under conditions simi‐ lar to iodoacetamides and maleimides to form stable mercury-thiol bonds that can be re‐ versed in 0.1N HCl and reducing reagents like dithiothreitol (DTT) but apparently not by TCEP. Phenylmercury derivatives also react faster with thiols than do the commonly used thiol-reactive *N*-ethylmaleimide (NEM).

Compounds containing disulfide bonds are able to undergo disulfide exchange reactions with another thiol by the free thiol attacking the disulfide bond and subsequent formation of a new mixed thiol. Two examples of useful compounds in this category are Methylmethane‐ thiosulfonate (MMTS) and pyridyl disulfide derivitives like biotin HPDP ((*N*-(6-(Biotinami‐ do)hexyl)-3'-(2'-pyridyldithio)-propionamide). MMTS can be used in some cases to block free thiols more effectively than NEM, as it is uncharged and thus more likely to modify all free reactive cysteines. MMTS has been shown not to react with nitrosothiols or existing di‐ sulfides [121].
