Foreword: Protein acylation and microdomains in membrane function

Protein acylation, the covalent attachment of long chain fatty acids to proteins, has been known for about 30 years but is currently enjoying a heyday, which is highlighted in this thematic issue. S-acylation (also called thioacylation or palmitoylation), involving thioester linkage of the fatty acid to protein cysteine residues, is of particular interest because it has the unique property among protein lipid modifications that it is reversible in vivo under physiological conditions. Several major recent advances have contributed to the explosion of studies in this field: the development of new assays, the identification of enzymes catalyzing acylation and deacylation, and increased understanding of the functions of protein acylation. The chapters below, by leaders in all aspects of the area, provide a snapshot of the current state of the art in this burgeoning field.

The first paper by Draper and Smith summarizes new assays for protein acyltransferases which have increased the speed and sensitivity of detecting the modification and substantially superceded the need for radioactive palmitate incorporation, although this still has its place as the gold standard method. The field of protein acylation has also suffered from the lack of specific and potent inhibitors, previously relying on rather unsatisfactory compounds such as 2-bromopalmitate and cerulenin that have multiple side effects. Given the great potential for therapeutic intervention targeted at protein acylation in several diseases, these inhibitors could form the basis for useful drugs in future; Draper and Smith present recent studies on new lead compounds that specifically target protein S-acyltransferases (PATs).

A massive boost to the field was given in the early 2000s when, after years of unsuccessful attempts by many groups, DHHC-CRD family PATs were identified by Deschenes’ and Linder's groups first in yeast [1] and subsequently in vertebrates. There are currently seven yeast DHHC-CRD PATs known and 23 in man, which show both partial redundancy and specificity in their substrate repertoire. This family of enzymes is reviewed by Planey and Zacharias who also highlight novel acylation assays and how they are being used to study the palmitoylproteome of different organisms and cell types.

For S-acylation to be reversible, protein acylthioesterases are required. This small and understudied family of enzymes is reviewed by Zeidman, Jackson and Magee in the third paper. In contrast to the great diversity of DHHC PATs, only two protein acylthioesterases have definitively been shown to deacylate intracellular proteins under conditions where they could play a role in physiological regulation of the acylation-deacylation cycle. Does this mean that all the specificity in these cycles comes from the PATs and the thioesterases are simply broad specificity enzymes that catalyse deacylation of a wide range of substrates? Too little is currently known about these enzymes to answer this question, but an intriguing possibility springs to mind from the observation of Zeidman et al. that these proteins, which are inherently hydrophilic in nature, show a substantial fraction associated with cellular membranes. One wonders whether they could associate with specific subcellular membranes through dedicated adaptor and targeting molecules, in an analogous fashion to the targeting of protein kinases A and C by A kinase anchoring proteins (AKAPs) and receptors for activated C kinase (RACKs) respectively [2], [3].

The next paper entitled ‘Palmitoylation cycles and regulation of protein function’ by Kanaani and Baekkeskov elegantly illustrates how acylation-deacylation cycles can modulate protein localization and function. Many acylated proteins are inherently soluble based on their primary sequence and their membrane localization is often induced by the attachment of fatty acids. However, many transmembrane proteins are also acylated and in this case the lipidation is not responsible for membrane attachment but has subtle effects on protein folding, assembly, intracellular trafficking and function, as eruditely described by Charollais and van der Goot. One of the interesting features brought out in this paper is how acylation of membrane-proximal regions can change the orientation of transmembrane domains with respect to the membrane and affect protein function.

One of the best characterized functions of protein acylation is targeting to specialized membrane subdomains, often but controversially called lipid rafts [4], reviewed by Chamberlain and colleagues. Greaves et al. describe how acylation acts as a protein sorting signal, affecting membrane trafficking, and protein stability by working contrarily to ubiquitination.

The next two papers focus on specific acylated proteins and their roles in signalling in the context of the Ras pathway and T cells. Henis et al. summarize how Ras proteins are exquisitely compartmentalized into nanodomains on the inner surface of the plasma membrane, the complex regulation of Ras signaling mediated by this microlocalization, and how it is affected by the lipid composition of the membrane which provides specific lipid-protein interactions. This paper also illustrates beautifully how a combination of high resolution light and electron microscopy combined with mathematical analysis has been used to dissect these processes.

Protein acylation and localization in T cell signalling is thoroughly reviewed by Bijlmakers. Many proteins involved in T cell signalling, both transmembrane and peripheral, are acylated and the lipid modifications play significant roles in function, not least through localization to lipid raft domains which seem to play a particularly important role in signalling in T cells and other cells of the immune system. Bijlmakers highlights the point that identifying DHHC-CRD PATs for specific T cell proteins will be crucial in designing pharmacological agents to target them in human diseases such as inflammation and cancer.

The penultimate paper on ‘Acyltransferases for secreted signalling proteins’ by Chang and Magee switches emphasis from intracellular acylated proteins modified by DHHC–CRD PATs to extracellular proteins acylated by a different family of enzymes. Members of the MBOAT family are responsible for acylation of important secreted signalling molecules involved in development, tumourigenesis and appetite control. These multispanning membrane enzymes acylate their substrates in the lumen of the secretory pathway, thus providing an interesting topological problem as the cosubstrate palmitoyl coenzyme A is thought to be in the cytoplasm. The study of protein acylation by these enzymes is at a very early stage and has interesting features, e.g. palmitic and palmitoleic acids can be added selectively in thioester or oxyester linkage, respectively, and in some cases a N-terminal amide-linked acylation can occur, probably by intramolecular rearrangement of a thioester. Again, this family of enzymes has great potential as therapeutic targets.

In the final paper, Hemsley reviews protein acylation in plants, which shows many parallels to the situation in animals. Higher plants contain a large number of DHHC-CRD PATs, plant S-acylated proteins include many signalling molecules, and the S-acylation machinery is also exploited by pathogens. S-acylation should provide rich veins of study both for basic and applied science in the plant kingdom.

In summary, S-acylation is enjoying a resurgence of interest both as a source of fascinating biology, but also offering great potential for practical applications in human, animal and plant disease. This issue will provide surprises even for experienced researchers in this area (as I found out when reading the contributions!) but will also stimulate the interest of newcomers to the field and hopefully induce them to enter this fascinating area of biology.