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

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 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‐ low but NEM is used most commonly.

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

Discovery of Selective and Potent Inhibitors of Palmitoylation

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

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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

like this always follow.

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‐ mine.

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‐ yl proteins, including PSD-95 and SNAP-25, when compared to labeling with 3 H-palmitate. 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‐ ginning to materialize.
