Exploring protein lipidation with chemical biology.

Protein lipidation is the covalent attachment of a lipid group to protein. Lipids modify large numbers of eukaryotic proteins and regulate protein function and localization. The hydrophobic character of lipid modifications makes the study of protein lipidation challenging. Chemical biology has played an increasingly important role in advancing the field of protein lipidation through novel methods of detection and isolation, design and synthesis of inhibitors, strategies to monitor the behavior of lipidated proteins in cells, and methods to produce lipidated proteins for structural and biophysical analyses. In this review, we discuss the chemical tools that have been developed to facilitate discovery in the field of protein lipidation and provide examples of how this has created new understanding of the scope of protein lipidation and its biological consequences. The content of this review is limited to the major posttranslational modifications that occur in the cytoplasm or on the cytoplasmic face of membranes: S-prenylation, N-myristoylation, and S-palmitoylation (Fig. 1). Protein lipidation of molecules destined for secretion occurs in the lumen of organelles within the secretory pathway. Glycosylphosphatidylinositol (GPI) anchors attached to proteins in the endoplasmic reticulum tether proteins to the extracellular face of the plasma membrane. Other secreted proteins that are lipidated include morphogens, cytokines, and hormones. Examples include the octanoylated peptide hormone ghrelin and the Hedgehog signaling protein, which is modified with both cholesterol and palmitate. Lipid modifications that occur in the lumen of secretory organelles are reviewed elsewhere. 1 FIGURE 1 Survey of cytosolic protein lipidation in eukaryotes. A) S-Farnesylation. B) S-Geranylgeranylation. C) C-phosphatidylethanolaminylation. D) N-myristoylation. E) N-palmitoylation. F) S-palmitoylation. 2. Lipid Modifications on the Cytoplasmic Face of Membranes Fatty acids and isoprenoids anchor proteins to the cytoplasmic leaflet of cell membranes (Fig. 1). Protein S-prenylation and N-myristoylation are mediated by cytoplasmic enzymes, whereas S-palmitoylation occurs at the membrane surface. Two other lipid modifications that occur in the cytoplasm are more rare. Amide-linked palmitate (N-palmitoylation) is found on the amino terminus of the signal transducing protein Gsα (Fig. 1E)2; phosphatidylethanolamine is linked to the C-terminal glycine residue of LC3/Atg8, a protein involved in autophagy (Fig. 1C) 3. In this section we briefly review the structure and mechanism of the major lipid modifications. S-Prenylation refers to the irreversible addition of an isoprenoid, either C15 farnesyl (Fig. 1A) or C20 geranylgeranyl (Fig. 1B), to protein through a thioether linkage.4 S-Prenylated proteins are recognized by their characteristic C-terminal motifs. Farnesyl transferase (FTase) and geranylgeranyltransferase type I (GGTase-I) modify proteins at a Cys residue that is four amino acids from the C terminus (the -CaaX box, where ‘a’ is a small, typically aliphatic amino acid and the identify of X contributes to the specificity of the prenyl group added). S-prenylation is followed by the proteolytic removal of the three terminal amino acids (–aaX) by Ras converting enzyme 1 and carboxylmethylation of the S-prenylated cysteine by isoprenylcysteine methyltransferase.5 Members of the Rab family of GTPases are prenylated by a dedicated enzyme, Rab geranylgeranyltransferase (RGGT).6 Rab proteins terminate in –CC, –CCXX, or –CXC motifs and are typically modified with two geranylgeranyl groups. Proteins modified with prenyl groups require the modification for membrane association and consequently to exert their functions.4 The realization that S-prenylation is essential for proper protein function has stimulated the development of small molecule S-prenyltransferase inhibitors to block the activity of oncogenic Ras and other S-prenylated proteins that malfunction in various disease states.7 Protein N-myristoylation refers to the irreversible addition of myristic acid to eukaryotic and viral proteins through an amide linkage to an N-terminal glycine residue (Fig. 1D).8 Modification of the protein typically occurs cotranslationally as the nascent polypeptide chain emerges from the ribosome and following removal of the initiator methionine by methionylpeptidase. Alternatively, during apoptosis, N-myristoylation can occur posttranslationally on proteins where caspase cleavage exposes a cryptic N-terminal glycine residue.9 The enzyme responsible for myristoylation is N-myristoyltransferase (NMT).8 Loss of NMT function in S. cereviseae is lethal10 and Drosophila lacking NMT have multiple developmental defects11. Thus, like S-prenylation, N-myristoylation is an essential process. Humans and mice have two N-myristoyltransferase genes, NMT1 and NMT2, that encode enzymes with overlapping functions. NMT1 is required for early embryonic development in mice.12 Proteins are fatty acylated at cysteine residues through reversible thioester linkages in a process commonly referred to as S-palmitoylation (Fig. 1F).13 Thioacylation or S-acylation is a more appropriate term for this modification given that other long chain fatty acids besides palmitate are incorporated into proteins through this mechanism. Both integral and peripheral membrane proteins can be modified with palmitate and this occurs both on intracellular membranes and at the plasma membrane. Promoting membrane association of soluble proteins is an obvious function for S-palmitoylation, but this modification also regulates protein trafficking, stability, and activity.14 The reversibility of protein S-palmitoylation distinguishes it from other lipid modifications and predicts the existence of enzymes that palmitoylate and depalmitoylate proteins. Protein S-acyltransferases (PATs) that modify proteins on the cytoplasmic face of membranes were first characterized molecularly in 2002.15 These are a family of integral membrane proteins that share a protein domain referred to as the DHHC domain, a cysteine-rich domain with a conserved Asp-His-His-Cys signature motif that is required for enzymatic activity. Yeast have seven DHHC-PATs; genetic and biochemical analyses have revealed that these enzymes can account for nearly all palmitoylation events in yeast.16 A larger family of 23 DHHC enzymes is found in mammals.17 The process of depalmitoylation is less well characterized. The lysosomal enzyme responsible for degradation of S-palmitoylated proteins may be protein palmitoylthioesterase 1 (PPT1).18 A cytoplasmic enzyme, acylprotein thioesterase 1 (APT1) removes palmitate from G-protein alpha subunits and Ras proteins in vitro.19 In vivo, APT1 has been linked to G-protein depalmitoylation in yeast20 and to Ras depalmitoylation in mammalian cells.21 The development of small molecule APT1 inhibitors has provided an important new tool to investigate the functional consequences of reversible palmitoylation of Ras.21 These are discussed in section 6. S-palmitoylation is often found in combination with other lipid modifications.14 Proteins with Cys residues adjacent to or near to an N-myristoylated Gly are often S-palmitoylated. Signal-transducing proteins such as G-protein α subunits22 and non-receptor tyrosine kinases23 are examples of proteins that are modified in this way. Similarly, S-prenylated proteins with cysteine residues immediately upstream of the S-prenylated cysteine are also substrates for S-palmitoylation. N-Ras and H-Ras are modified with palmitate at one or two cysteines, respectively. Early studies revealed that N-myristoylation or S-farnesylation was a requirement for subsequent S-palmitoylation and that both the myristoyl or farnesyl modification and S-palmitoylation were necessary for stable membrane association and plasma membrane localization. As discussed in the following sections, the development of synthetic lipopepides and semisynthesis of lipidated proteins was key to understanding the physical basis of the membrane interactions and the functional significance of reversible S-palmitoylation.