# Structural Biochemistry/Proteins/Palmitoylation

Palmitoylation is the covalent attachment of deprotonated fatty acid palmitic acid to a cysteine residue on a membrane protein. Palmitoylation has many different functions in proteins. For instance, it functions as membrane attachment regulation, intracellular trafficking, and membrane micro-localization.
Specifically, S-palmitoylation refers to the reversible thioester linkage, or bond between the sulfide on the cystine to the electron rich oxygen of the palmitate. N-palmitoylation is similar, however, when the cystine is located on the N-terminus of the protein, the palmitate temporarily bonds with the sulfur, like in S-palmitoylation, and then quickly binds to the amide of the cystine for stability. (4)
Palmitoylated cystines share a few common features that narrow which cystines will and will not be palmitoylated such as: they are located in the cytoplasmic region, exposed in a peptide sequence so that they can be easily palmitoylated; they are sequenced next to or closely by hydrophobic or basic amino acids; they are often, but not exclusively, adjacent to prenylation and myristoylation sites. (5)
Once a membrane protein contains a palmitate, the membrane protein can interact with other lipids and proteins in new ways, effectively altering some of the proteins functions such as intracellular sorting, membrane interactions, stability, and membrane micropatterning. (5) Palmitoylation enhances the hydrophobicity of proteins and contributes to their membrane association. Interestingly, S-palmitoylation is a reversible process, which allows for unique and complex modes of trafficking between membrane compartments. (4) This is in contrast to prenylation and myrisoylation, which are not irreversible. The specific function of palmitoylation depends on the particular protein being considered.
Palmitoylation also appears to play a significant role in subcellular trafficking of proteins between membrane compartments, as well as in modulating protein-protein interactions. The specific location of where palmitoylation occurs gives insight into what proteins are palmitoylated and for what reasons. A location with high amounts of palmitoylation occurring takes place near the golgi, where peripheral membrane proteins are palmitoylated. In post golgi compartments, specific substrates are palmitoylated for the purpose of regulation. (5)

## Palmitoylation in Action

An example of a protein that undergoes palmitoylation is hemagglutinin, a membrane glycoprotein used by influenza to attach to host cell receptors.
Since S-palmitoylation is a dynamic, post-translational process, it is believed to be employed by the cell to alter the subcellular localization, protein-protein interactions, or binding capacities of a protein.
The palmitoylation cycles of a wide array of enzymes have been characterized in the past few years, including H-Ras, Gsα, the β2-adrenergic receptor, and endothelial nitric oxide synthase (eNOS).
Another example of proteins that undergo palmitoylation is the cell's ion channels. Throughout an ion channels life cycle, palmitoylation plays a crucial role in controlling and regulating its signaling pathways. From assembly of the ion channel to the recycling and degradation of the it, palmitoylation is controlled by the family of acyl palmitoyltransferases and a particular number of thioesterases. The NMDA receptors are palmitoylated at the golgi to keep these proteins anchored to the goli whereas palmitoylation of calcium- and voltage-activated potassium channels contributes to their cell-surface delivery. This demonstrates how dynamic and unique palmitoylation is, occurring at the same location for different proteins but with considerably different results. (6)

## Palmitoylation in Synaptic Plasticity

Recently, scientists have appreciated the significance of attaching long hydrophobic chains to specific proteins in cell signaling pathways. A good example of its significance is in the clustering of proteins in the synapse. A major mediator of protein clustering in the synapse is the postsynaptic density (95kD) protein, PSD-95. When this protein is palmitoylated it is restricted to the membrane. This restriction to the membrane allows it to bind to and cluster ion channels in the postsynaptic membrane. Also, in the presynaptic neuron, palmitoylation of SNAP-25 allows the SNARE complex to dissociate during vesicle fusion. This provides a role for palmitoylation in regulating neurotransmitter release.

## DHHC Palmitoyl Transferases

Internal cellular palmitoylation reactions are monitored by a palmitoyl transferase known as aspartate-histidine-histidine-cysteine, or more commonly known as DHHC. Researches have indicated that there are approximately 20 DHHC proteins that are present in mammalian genomes and have proven that they are crucial in cell function and physiology and pathophysiology influence.
Studies regarding yeast have established interesting characteristics of the DHHC protein family. For one, some DHHC proteins require protein cofactors. Second, DHHC play a very important role in cellular activity. Moreover, DHHC palmitoyl transferases are mobile to specific intracellular membranes. More precisely, the DHHC-CR domain is a prominent feature of palmitoyl transferases, and genes that are associated with DHHC proteins are found in organisms from yeast to humans. Approximately seven DHHC proteins are relevant for yeast organism, whereas more than 20 DHHC genes are discovered for mammalians. Most of the mammalian DHHC proteins have the tendency to catalyze palmitoylation independently of other protein cofactors.
There is not sufficient amount of data that has proven that DHHC proteins have achieved their respective intracellular localizations. However, a general scope of analysis has shown that a removal of a phenylalanine residue in the C terminus of DHHC21 repositions the proteins from the Golgi to ER membranes. Furthermore, mutations that occur at the 16-amino acid motif in the C terminus of DHHC proteins of yeast organisms cause a mis-localization from the vacuole membrane to the lumen. Protein degradation is based on the yeast vacuole functionality in the sense that there is consistency in the decreasing expression levels of mutant protein. The analysis of DHHC proteins and their presence in cellular palmitoylation can be seen in the systematic experiment of S.cerevisae where a proteomic investigation of substrate palmitoylation profiles in deficiency in DHHC yeast strains imply a substrate selectivity. For instance, depletion in Akr1 led to an enormous downfall in palmitoylation of Yck1 and Yck2. Furthermore, it caused a removal of palmitoylation of Meh11, Yp199c, and Yk1047w. Conclusively, this experimental study has underlie that individual yeast DHHC proteins express a preference towards specific substrates Akr1 target were soluble proteins that are exclusively palmitoylated; Erf2 substrates had a tendency to be modify by myristoyl or prenyl groups; and Swf1 contained a preference for cysteine residues.
Protein binding assays have determined locations of palmitoylated substrates that communicate with DHHC proteins. Studies have shown that short, minimal sequences from palmitoylated proteins process effective palmitoylation when expressed in cells. Even though yeast genetic studies have indicated characteristics that substrates are affiliated with the modifications of DHHC protein, this cannot be said about mammalian proteins. This is due to the fact that there is a lack of data that show the substrate interactions performed on mammalian DHHC substrates. Thus, DHHC protein-substrate specificity is a subject that is hard to generalize and contain many undeveloped areas of research.

## References

1. Linder, M.E., "Reversible modification of proteins with thioester-linked fatty acids," Protein Lipidation, F. Tamanoi and D.S. Sigman, eds. (San Diego, CA: Academic Press, 2000).
2. Basu, J., "Protein palmitoylation and dynamic modulation of protein function," Current Science, Vol. 87, No. 2, (25 July 2004).
3. Smotrys J and Linder A. (2004) "Palmitoylation of Intracellular Signaling Proteins: Regulation and Function". Annu Rev Biochem 73.
4. Linder, M. and Deschenes. R., "Palmitoylation: policing protein stability and traffic," Nature Review (January 2007)
5. Salaun, C et al., "The inracellular dynamic of protein palmitoylation," J. Cell Biol. Vol. 191 No.7 (December 2010)
6. Shipston MJ, "Ion channel regulation by protein palmitoylation," J Biol Chem. 2011 March 18; 286(11): 8709–8716 (January 2010)
7. Greaves, Jennifer, Luke H. Chamberlain, DHHC palmitoyl transferases: substrate interactions and (patho)physiology, Trends in Biochemical Sciences, Volume 36, Issue 5, May 2011, Pages 245–253, ISSN 0968-0004, 10.1016/j.tibs.2011.01.003. (http://www.sciencedirect.com/science/article/pii/S0968000411000144)