Proteolipids and Protein Lipidation

In 1951, proteins that were soluble in organic solvents such as chloroform-methanol were found in rat brain myelin by Folch and Lees (perhaps better known for devising the most common method for lipid extraction), who coined the term ‘proteolipid’, but it was a further twenty years before it was shown that these contained covalently bound fatty acids and so differed from the plasma lipoproteins. Such lipid-modified proteins, formed in post-translational reactions, are now known to be widespread in nature with many vital functions in animals, plants and bacteria. Proteolipids can be defined as all proteins containing covalently bound lipid moieties such as fatty acids, isoprenoids, cholesterol and glycosylphosphatidylinositols (the last have their own web page). Protein lipidation provides an interface between the lipophilic membranes of cells and otherwise hydrophilic proteins that enables signalling and many other activities at the membrane surface; dysregulation impacts upon physiology and disease. Here, proteolipids from animals and plants are discussed, but bacterial lipopeptides and proteolipids are sufficiently different to have their own web page. The term ‘lipoprotein’ is sometimes used to describe such compounds on occasion, but to avoid confusion this might be better reserved for the non-covalently linked lipid-protein complexes of the type found in plasma.

1.  Introducing Protein-Lipid Modifications

It is a curious but noteworthy fact that the two main types of protein with long-chain fatty acid modifications to have been described from eukaryotic organisms contain saturated fatty acid components, i.e., those with only myristoyl and those with predominantly palmitoyl moieties, each with a distinctive type of linkage, amide or thiol ester, respectively, although a few O‑acylated proteins are known. These modifications can be differentiated by the main amino acid residues (glycine, cysteine or serine) that are involved. Prenylated lipids contain an isoprenoid group linked via a sulfur atom (thiol ether bond) to the protein. Often, N‑myristoylated and prenylated proteins have one or more additional S-acylation residue. Covalent modification of proteins with lipids has the effect of changing them from a generally hydrophilic nature to one that is hydrophobic at one end at least, thus facilitating the interaction with membranes.

Figure 1. Formulae of myristoylated, palmitoylated and prenylated proteins

Protein S-palmitoylation occurs post-translationally at the membrane surface, while N‑myristoylation takes place at the ribosomal level, and S-prenylation is brought about by cytoplasmic enzymes. It is now clear that such modifications can determine the activities of proteins and target them to specific subcellular membrane domains, including the rafts and caveolae in plasma membranes. Thus, both myristoylated and palmitoylated proteins are targeted to rafts (as are the GPI-anchored proteins), but prenylated lipids are not. It is significant that many signalling proteins, e.g., G-protein-coupled receptors and protein tyrosine kinases, and often their substrates are modified by lipids with implications for the relevant signalling events at the cell surface. Of these, S-palmitoylation is readily reversible so has drawn comparisons with such modifications such as phosphorylation and ubiquitination.

Figure 2. Protein acylation forms

Other protein-lipid links exist and are discussed below, but they tend to be less common. One class of proteolipids contains a linkage to cholesterol as well as N‑palmitoylation, the ‘hedgehog’ signalling proteins, while bacterial proteolipids contain N-acyl- and S‑diacylglycerol groups attached to an N-terminal cysteine.

Such proteolipids are critical for all classes of eukaryotes (animals, plants and fungi) and bacteria, and they many influence human disease states and are potential pharmacological targets. Deregulation of palmitoylation has been associated with heart disease, cancer, mental retardation and schizophrenia, while some pathogenic organisms can hijack the protein acylation mechanism to increase the susceptibility of the host to infection.

2.  N-Myristoylated Proteins

In most N-myristoylated proteins, myristic acid (14:0), a ubiquitous but usually minor component of cellular lipids (only 1% of the total fatty acids), is bound to the amino-terminal glycine (rarely cysteine) residue of a relatively conserved sequence of the protein via an amide linkage that is comparatively stable to hydrolysis. Therefore, for most proteins, N-myristoylation in this way is an irreversible stable modification in essence, and the half-life of a myristoylated protein is like that of the nascent polypeptide chain. These proteolipids constitute a large family of eukaryotic proteins (~2% of the total) with many different functions, and they are located either in the cytosol or in the cytosolic (inner) membrane of cells, or both. Indeed, N-glycine myristoylation mediates the targeting of the modified proteins to various membranes in the cell, including the plasma membrane, endoplasmic reticulum, Golgi, mitochondria and nuclei. The acyl group anchors the protein to membranes, although simultaneous binding to phospholipids or other membrane constituents increases the strength of the interaction. As a single N‑myristoylation is not sufficient to ensure membrane association, a second binding site is necessary, e.g., hydrophobic residues, another membrane-bound binding partner or further N‑myristoylation or S‑palmitoylation (see below).

During biosynthesis, the leader methionine residue is first removed from the nascent peptide chain by a methionine aminopeptidase (MAP) to expose the N‑terminal glycine before an N‑myristoyl-CoA:protein N-myristoyltransferase (NMT1 or NMT2 in humans) catalyses the formation of the stable amide bond as it begins to emerge from the ribosome, i.e., it is mainly a co-translational rather than a post-translational event. Myristoyl-CoA is supplied by an acyl-CoA binding protein (ACBD6), and this together with the size of the NMT binding pocket controls the specificity of the reaction by forming a complex with the transferase thereby stimulating it and preventing it from utilizing the competitor palmitoyl-CoA. Most eukaryotes have such enzymes but not prokaryotes, although lower eukaryotes have only one isoform.

N-Myristoylation of proteins

While NMT1, but not NMT2, is utilized for cell proliferation, both are required for cell survival. NMT1 is located mainly in the plasma membrane while NMT2 is cytosolic (nearly 77% peptide sequence identity), and both have an N‑terminal region with polybasic amino acid sequences, which target proteins to the ribosomes to facilitate the co-translational timing of the reaction. They have partial substrate overlap but differential roles during embryogenesis and tumour cell proliferation. The crystal structure of NMT1 has shown that it has a characteristic binding cleft that is involved in the recognition of potential substrates for myristoylation (with some overlap with targets for N-acetylation), and there is an absolute requirement for the N-terminal glycine on the target protein with amino acid residues two through eight as the consensus motif (Met-Glc-X-X-X-Ser/Thr/Cys) used in substrate recognition.

Exceptions to these generalities are photoreceptor proteins, which are modified heterogeneously with the uncommon 12:0, 5‑14:1 and 5,8‑14:2 fatty acids as well as 14:0, and certain histone proteins can be modified by various fatty acids. In the model nematode Caenorhabditis elegans, N-terminal glycine is acylated almost exclusively with myristic acid, but lysine (see below) is acylated preferentially with isomethyl-branched-chain fatty acids. N-Palmitoylated proteins have been found on occasion, usually where there is a dual lipid modification as with the cholesterol-linked hedgehog proteins (see below). As a further exception, it has become apparent that myristoylation can occur post-translationally in apoptotic cells on internal glycine residues exposed by caspase cleavage in partially hydrolysed proteins, a process that may have implications for health and disease.

N-myristoylated proteins with dual modifications can be dissociated from membranes by de‑S‑palmitoylation, by phosphorylation, by binding of the hydrophobic moieties to competing cytosolic proteins (if the myristoyl moiety is relatively exposed) or by conformational changes, and these reactions are forms of regulation. The low levels of myristic acid in tissues may be a further controlling factor. Macrophages contain at least one protease that targets certain N‑myristoylated proteins, and similar proteases must operate in other tissues. On the other hand, myristoylation promotes protein stability by inhibiting the proteasome degradation pathway.

Scottish thistleIn humans, 569 proteins are now known to be myristoylated, and these include proteins that have roles in signalling networks, apoptosis, oncogenesis and viral replication, all of critical physiological importance. The endoplasmic reticulum-Golgi network and the plasma membrane are the main targets of myristoylated proteins, but mitochondria, cilia, nuclei and the endolysosomal network are targeted in the same way. Their lipid moieties are involved in regulating protein efficiency perhaps by modifying or stabilizing their conformations, by facilitating protein-protein interactions, and in targeting otherwise soluble proteins to the membranes and to appropriate receptors. N‑Myristoylation is essential for erythrocyte formation and myelination, it is necessary for calcium release in mitochondria, and it is a factor for T cells and the immune response. During apoptosis, NMTs are substrates for caspases, which are believed to regulate their location from the ribosomal and membrane regions to the cytosol and vice versa.

Increased levels of N-myristoylation have been observed in certain cancers, and there have been suggestions that NMTs could be a target for therapeutic intervention. NMT is believed to play a part in innate immunity, the evolutionarily conserved host defence mechanism that responds immediately to pathogens but does not provide long-lasting immunity to the host (in contrast to adaptive immunity). It is relevant to infections by viruses, including human immunodeficiency virus-1 (HIV-1), bacteria, e.g., Shigella flexneri, fungi and parasitic protozoa, including such diseases as malaria. The Shigella virulence factor IpaJ has been identified as an irreversible demyristoylase.

Plants: 650 Arabidopsis thaliana proteins are potentially myristoylated, including many protein kinases, phosphatases, thioredoxins and transcription factors. As an example, the calcium-sensing molecule CBL1 uses N-myristoylation for association with the endoplasmic reticulum followed by S‑acylation to promote trafficking to the plasma membrane; in contrast CBL2 is not N-myristoylated but rather is triply S-acylated to direct it to the tonoplast.

Lysine fatty acylation: It is now recognized that a second form of protein N-acylation exists in which lysine residues are acylated (mainly with 14:0), again with the effect of increasing the affinity of proteins for specific cellular membranes, although so far, a relatively limited number of proteins are known to be modified in this way including tumour necrosis factor α (TNF-α), interleukin 1-α and some of the Ras family of GTPases. With these, different enzymes or iso-enzymes from those for N‑terminal myristoylation are involved, the reaction occurs by a somewhat different mechanism and is inefficient. In this instance, the modification is reversible as sirtuins 2 and 6 (SIRT2/6), mammalian nicotinamide adenine dinucleotide (NAD)-dependent lysine deacylases, catalyse the removal of fatty acyl groups from lysine residues, and they are thus able to regulate the relevant proteins. Demyristoylation at two sites of gravin-α in adipocytes by the enzyme histone deacetylase 11 (HDAC11) prevents β-adrenergic (G protein-coupled) receptors from translocating to the membrane microdomains that are utilized for downstream protective signalling, in effect a control mechanism with relevance to obesity. Among reported ill effects of lysine fatty acylation are tumorigenesis and increased pathogenesis of bacteria. In plants, N-acylation of lysine residues with various phytohormones, including jasmonic acid, has been reported. N-acetylation of lysine residues is discussed below.

Lipoic acid is a cofactor for cofactor for five enzymes or classes of enzymes, two of which are critical to the citric acid cycle. It is synthesised from octanoic acid transferred as a thioester of acyl carrier protein from fatty acid biosynthesis to a terminal lysine residue of the enzymes' lipoyl domains where it is linked by an amide bond; it is only released when the enzymes are eventually degraded.

Analysis: Mass spectrometry is now widely used for characterization of N-myristoylated proteins, although the fatty acyl group can be released for analysis by conventional chromatographic means (e.g., gas chromatography) by the acidic hydrolysis conditions commonly employed to cleave peptide bonds. Treatment with 6M HCl or 2M HCl in 83% methanol at 100°C for several hours is needed to release the N-acyl group as the free fatty acid or methyl ester, respectively. New methods involving specific chemical probes and mass spectrometry are now facilitating detection and analysis of N‑myristoylated proteins and other proteolipids.

3.  S-Acylated (Palmitoylated) Proteins

In the S-acylated proteins, long-chain fatty acids are linked to one or more (up to six) internal cysteine residues via labile high-energy thioester bonds. As palmitic acid (16:0) is the most common acyl component, these are often termed S-palmitoylated proteins, but this is something of a misnomer as other fatty acids are often present, including 16:1, 18:0 and 18:1 (one screen found 218 proteins acylated with 16:0 and 308 proteins with C18 fatty acids in humans), while S-18:0 is as common as S-16:0 in plants, and viral proteins are often linked to C18 fatty acids; in the nematode C. elegans, 15‑methylhexadecanoic acid is preferred. This may simply reflect the relative availabilities of these fatty acids at each acylation site. There is some evidence that the nature of the acylating fatty acid can affect the efficiency of the resulting proteolipid, so the term 'S-acylation' is more accurate. In contrast to N-myristoylation and isoprenylation, there does not appear to be any specific peptide target sequence, although proteomic methods now enable prediction of which cysteine residue is likely to be palmitoylated. It is now apparent that protein palmitoylation is essential for intracellular signalling and for the folding, trafficking and function of such disparate proteins as Src-family kinases, Ras family GTPases, G proteins and G protein-coupled receptors. As S-acylation is reversible by deacylation, it is in effect an on/off switch, analogous to phosphorylation, acetylation and ubiquitylation.

The archetypal proteolipid protein (‘PLP’) or lipophilin is the main protein in the myelin of the central nervous system and was the first of its type to be identified and properly characterized. It is a highly conserved hydrophobic protein of 276 to 280 amino acids with four transmembrane segments that binds at least six palmitoyl groups via thioester bonds. A wide variety of different palmitoylated proteins are now known, and this is most prevalent type of protein modification by lipids with more than 500 different palmitoylated proteins identified in humans (10-20% of the proteome), 1094 in A. thaliana (6% of the proteome) and over 50 in the yeast Saccharomyces cerevisiae. These can be grouped into three broad categories - poly-acylated membrane proteins (e.g., some receptors and rhodopsin), mono-acylated membrane proteins (some receptors and viral proteins) and hydrophilic proteins (such as certain protein kinases). In brain, 490 palmitoylation sites have been identified on 342 synaptic proteins, 44% of which are integral membrane proteins. A comparison of orthologues between such distant phyla as mammals and Drosophila melanogaster suggests that S-palmitoylated proteins are more conserved than are non-S-palmitoylated proteins.

A zDHHC protein in a membraneThio-acylation: The reaction occurs post-translation of the protein and is catalysed by specific endomembrane-bound acyltransferases at specific cysteine residues exposed to the cytosol. Although there is some evidence for occasional non-enzymatic palmitoylation, enzymatic mechanisms predominate, and palmitoyl S-acyltransferases were identified definitively first from yeasts and subsequently from mammalian cells. Families of such enzymes (23 in humans coded by more than 20 separate genes, 24 in mice, a similar number in Arabidopsis and 7 in yeasts) have now been characterized with a conserved cysteine-rich domain containing zinc and a distinctive aspartate-histidine-histidine-cysteine motif (DHHC, or ZDHHC in humans and zDHHC in mice) within a cysteine-rich region, where the catalytic reaction occurs. They are membrane proteins with many subcellular locations that span the bilayer at least four times, with the DHHC domain and the N- and C‑terminal domains on the cytosolic face. In most palmitoyl transferases, there is a conserved C‑terminus (PaCCT) domain, and this is required for their function. With human DHHC20, it has been established that the fatty acyl chain of palmitoyl CoA inserts into a hydrophobic pocket within the transmembrane spanning region of the protein, while the CoA headgroup is recognized by the cytosolic domain through polar and ionic interactions.

Each of the mammalian DHHC proteins appear to be associated with specific subcellular locations but mainly the endoplasmic reticulum and Golgi, with a few at the plasma membrane and some at more than one location, often with some preference for particular tissues. Simplistically, Golgi-located palmitoyl acyltransferases promote the palmitoylation and forward trafficking of substrates to the plasma membrane, while those in other organelles tend to generate the products for local use. In plants, many of the enzymes are located at the plasma membrane.

DHHC acyltransferases operate by a two-step mechanism in the first step of which the enzymes are auto-acylated by a nucleophilic attack of the active site cysteine on the thioester of palmitoyl CoA with generation of an acyl-enzyme intermediate and release of CoA. In the second step, the acyl group is transferred to a protein substrate in a trans-acylation reaction. Cysteine palmitoylation forms a thioester bond that is similar in energy to that in the palmitoyl donor, palmitoyl-CoA, so the reaction does not need an energy source such as ATP. There is an absolute requirement for long-chain acyl-CoA esters, mainly 16:0, as fatty acyl donors, though some members of the DHHC protein family have different acyl specificities and at least one can use a wide variety of fatty acyl-CoA substrates. The acyl chain binds in a a lengthy cavity containing hydrophobic residues formed by the transmembrane domains, and the acyl chain-length selectivity is believed to depend upon cavity variants with preferences for specific acyl chains.

Protein palmitoylation-depalmitoylation

It is not yet fully understood how substrate recruitment and specificity are regulated for palmitoylation. One DHHC enzyme can often palmitoylate many different protein substrates, while a given protein may be palmitoylated by more than one acyl transferase. On the other hand, there are examples where specific proteins are palmitoylated by one DHHC enzyme. One common feature is that the active sites on the enzymes reside at the membrane-cytosol interface so that membrane-proximal cysteines are candidates for palmitoylation. While in general there does not appear to be a preferred substrate-sequence motif in target proteins, some DHHCs contain further sequences of amino acids (SH3 and Akr domains, PDZ binding motifs) that may be involved in enzyme-substrate recruitment. There is a suggestion that acyl-CoAs do not diffuse freely in the cytosol but are channelled into specific pathways by mechanism that may involve compartmentalization and intracellular transport. Some DHHCs must be palmitoylated by another before encountering their substrate protein. Prior to S-acylation, modification by N‑myristoylation or prenylation is often required, and with the former, specific target sequences for subsequent palmitoylation have been identified with further regulation by post-translational phosphorylation or ubiquitylation.

De-acylation: In contrast to irreversible N-myristoylation, hydrolysis of S-palmitoylated proteins occurs readily and is catalysed by thioesterases, two of which have been characterized in the cytosol, i.e., acyl protein thioesterase 1 (APT1 or LYPLA1) and 2 (APT2 or LYPLA2) each with specificities for particular classes of protein conjugates. Among other such enzymes, two lysosomal enzymes, palmitoyl protein thioesterases 1 and 2 (PPT1 & 2) and a mitochondrial enzyme (ABHD10) are known. All of these belong to the ‘metabolic’ serine hydrolase superfamily, with an active site serine for substrate hydrolysis and the ability to cleave ester, amide, or thioester bonds on many classes of molecules. Thus, most proteolipids of this type undergo cycles of acylation-deacylation, with a half-life (seconds to several minutes) that is much shorter than that of the peptide per se, and this permits proteins to shuttle between membranes and other cellular compartments, such as between the plasma membrane and Golgi in both directions. For example, after extracellular stimulation, Ras-proteins produce signals that lead to cell proliferation, differentiation and apoptosis; they are palmitoylated for transport to the plasma membrane then de-palmitoylated for transport back to the Golgi (in this instance by a family of ABHD17 serine hydrolases) so at the steady state, these proteins can signal at both organelles, a continual and essential cycle. Some proteins are prenylated and S-acylated in an active form, while the inactive form is only prenylated. Such acylation-deacylation cycles are believed to be regulatory, and synthetic and hydrolytic enzymes are themselves regulated dynamically by extracellular factors like phosphorylation, with the level of palmitoylation determined by a finely tuned balance between the enzymes in specific cellular locations.

Function: Scottish thistleAs with the myristoylated proteins, palmitoylation is believed to modify protein behaviour partly by modifying their conformations, but mainly in targeting otherwise soluble proteins to specific membranes or to appropriate receptors with the number of bound fatty acyl groups controlling the strength of the interaction with membranes. A hydrophobic protein with a single acylation can bind only loosely to membranes and is easily displaced, but a second or further acylation ensures strong targeting of a protein to the cytoplasmic face of the membrane and guarantees that it is firmly bound to a specific site where an appropriate receptor may be located. While many reported S-acylation substrates are already transmembrane proteins, acylation on a cytosolic brings it to the membrane.

Palmitoylation of integral membrane proteins may be regulatory, changing their conformation to increase their stability through protecting them from degradation by preventing ubiquitinylation and increasing their resistance to proteases. It is believed to be a factor in the process of trafficking proteins between organelles and in directing them to specific membrane compartments. In neurons, palmitoylation targets proteins for transport to nerve terminals and may regulate trafficking at synapses; it enables the growth and integrity of neuronal axons and is needed to convey axonal signals with effects upon learning and memory, and brain disorders associated with cognitive defects.

The saturated acyl moiety in palmitoylation facilitates transfer of proteins to lipid rafts, subdomains of the plasma membrane that are enriched in sphingolipids and cholesterol. Thus, S-palmitoylation enables flotillins to bind to the cytosolic leaflet of the plasma membrane in raft domains, where they oligomerize to serve as scaffolds that facilitate the assembly of multiprotein complexes at the membrane–cytosol interface. Proteins with many S-acylated cysteines near the membrane have the potential to induce formation of membrane nanodomains de novo through a phase-separation mechanism, and one such is the SARS-CoV-2 spike protein, which forms trimers and carries ten acylation sites per protomer. In association with specific nanodomains, proteolipids can participate in cell signalling events such as in T-cells of the immune system, where the unique feature of reversibility in lipid S-palmitoylation modifications is advantageous.

In general, S-palmitoylation stimulates signalling by the lipidated proteins. Protein S-palmitoylation is a basic mechanism for control of the properties and operation of ion channels, both directly and indirectly via other signalling pathways, and via activation of calcium channels in the plasma membrane and endoplasmic reticulum, this regulates apoptotic signalling. In the heart, S-palmitoylation is involved in the regulation of cardiac electrophysiology, including the sodium-calcium exchanger, phospholemman, the cardiac sodium pump and the voltage-gated sodium channel. Mitochondrial morphology and function are regulated by S‑stearoylation of human transferrin receptor 1. S‑Palmitoylation enables macrophages to respond appropriately in processes such as endocytosis, intracellular signalling, lysosomal sorting and chemotaxis. Among many further examples that could be listed, S-acylation regulates components of the insulin secretion and insulin response pathways, including channels, transporters and receptors.

Palmitoylation is involved in lipoprotein metabolism. Lipoprotein particles containing apolipoprotein B (apo B), such as chylomicrons, VLDL and LDL, transport triacylglycerols and cholesterol esters in plasma, and it has been established that palmitoylation of apo B regulates the biogenesis of the nascent lipoprotein particles that contain this apolipoprotein and may control the amount available for lipid transport.

Dysregulation of DHHC palmitoyl acyltransferases is known to be associated with many human diseases, including schizophrenia, X-linked mental retardation, Huntington's disease and cancer, and these enzymes are now seen as targets for drug development. Protein palmitoylation is crucial for many signalling pathways in tumours that affect their occurrence and progression and the therapeutic response, so that both palmitoylases and depalmitoylases are seen as potential targets for anti-cancer treatment. In relation to cardiovascular diseases, S-palmitoylation influences the function of cardiac myocytes by regulating ion channels and signal transduction and may be a factor in arrhythmia.

Scottish thistle Infections: The innate immune system responds to invasion by microorganisms by signalling through pattern recognition receptors (PRRs) that bind conserved microbial features. Many of these receptors, including the Toll-like receptors, are S‑palmitoylated, and this is believed to be a required modification for innate immune signalling.

Protein S-palmitoylation is critical for the pathogenicity of many human and plant pathogens, including fungal, bacterial and viral infections. While parasitic protozoa and fungi possess their own palmitoyltransferases, viruses and bacteria hijack the enzymes in their hosts to favour their internalization, survival and replication inside the cells. Palmitoylation of viral proteins is essential for their life cycle, and three types of membrane proteins in viruses, including many that are highly pathogenic, are known to be S-acylated, a factor that is necessary for the immune response. Palmitoylated 'spike' proteins are the main transmembrane proteins in the viral envelope, and they are involved in the entry of viruses into cells by catalysing receptor binding and/or membrane fusion. The viroporins are a second group, which are freely expressed in infected cells but not into virus particles per se to any appreciable extent; they possess one or two membrane-spanning regions, which serve as hydrophilic pores in membranes. A third diverse group of palmitoylated proteins produced by viruses are peripheral membrane proteins in which the fatty acid component simply anchors the modified protein to a membrane. In contrast to other proteins, glycoproteins of viral membranes are palmitoylated at or near the cytoplasmic face and then remain palmitoylated. S‑Palmitoylation is necessary for the survival, growth and infectivity of the trypanosomatids (protozoal parasites) in humans.

Plants: Numerous S-acylated proteins are known to be present in higher plants, and many are now being described, although progress appears to have been slower than with mammalian systems. As in animals, S‑acylation alters protein interactions with membranes together with their activation state, conformations and sub-cellular distributions, and the cycle of S-acylation and de-S-acylation provides a molecular mechanism for membrane-associated proteins to undergo cycling and trafficking between different cellular compartments. SNARE and G proteins and the cellulose synthase complex are among several proteins known to be modified in this way with effects on fertility, Ca2+ signalling, movement of potassium ions, stress signalling (e.g., the immune response), root hair and pollen tube formation, cell death, production of reactive oxygen species, cell expansion and division, gametogenesis and salt tolerance. The 18 subunits of the cellulose synthase A family have numerous S‑acyl groups (70–110), rendering it the most heavily S-acylated complex ever described. As this complex is integral to the plasma membrane and extrudes cellulose microfibrils into the extracellular environment to form the cell wall, the extrusion process is believed to enable the complex to pass through the plane of the plasma membrane to form microdomains and recruit accessory proteins for unhindered transport across the membrane.

Few S-acylated proteins have been matched to the any of the 24 S-acyl transferases in Arabidopsis, so each enzyme may have multiple substrates (as many as 100) involved in diverse processes. Protein thioesterases comparable to those in animals that reverse the reaction have not yet been characterized in plants, although two candidate enzymes have been identified that unexpectedly may not be in the serine hydrolase superfamily. S-Palmitoylation may be more important at the plasma membrane in plants than in animals.

Analysis: Modern mass spectrometric methods in concert with a site-specific acyl-biotin-exchange reaction permit location of the acyl group to specific cysteine residues. In contrast to the N-acylated proteins, the fatty acids are easily released from the thiol linkage by base-catalysed transesterification for analysis by gas chromatography.

4.  Prenylated Proteins

Prenylated proteins are formed by attachment of isoprenoid lipid units, farnesyl (C15) or geranylgeranyl (C20), via cysteine thio-ether bonds, which are more stable than ester or thioester bonds, at or near the carboxyl terminus. Such proteins were first detected in fungi, but they are ubiquitous in mammalian cells where they can amount to up to 2% of the total proteins, and they are increasingly being found in other eukaryotic organisms, while some proteins from pathogenic bacteria and viruses can be prenylated by their hosts, apparently as a protective measure. Isoprenylation is a stable (non-reversible) modification, which targets specific proteins to membranes, aids protein-protein interactions and enables their functions. In contrast to the acylated proteins, the bulky branched nature of the lipid moiety of isoprenylated proteins ensures that the latter cannot be incorporated into ordered raft microdomains.

Formula of a prenylated protein

Whether a protein is prenylated is determined by specific amino acid sequence motifs at the carboxyl terminus, principally a CAAX sequence with cysteine (C) attached to two aliphatic amino acids (A) then to a variable carboxyl-terminal amino acid residue (X). The nature of the X residue determines whether a protein will be farnesylated or geranylgeranylated; with a few exceptions, proteins ending in serine, methionine, alanine or glutamine are farnesylated, while proteins ending in leucine or isoleucine are geranylgeranylated. A single farnesylation is usual, but some dual geranylgeranylation can occur. Proteins involved in cellular signalling and trafficking pathways are involved mainly, and the best known of these are probably the Ras super-family (discussed above).

The isoprenoid units are produced by the mevalonate pathway, as discussed on our web page dealing with cholesterol with farnesyl pyrophosphate as the branch-point in sterol/isoprene synthesis. Subsequent biosynthesis of prenylated proteins involves a concerted series of reactions in which the proteins are transported through various cellular organelles, ending mainly but not only at the plasma membrane. Prenylation occurs in the cytoplasm of the cell after synthesis of the protein per se, with farnesyl or geranylgeranyl pyrophosphate as the isoprenoid substrate, each catalysed by its own transferases, four of which are known, i.e., protein farnesyltransferase and protein geranylgeranyl transferase (two types GGT-1 and 2), respectively. The enzymes transfer the isoprenoid group to the cysteine residue in the CAAX box, and enzyme-bound Zn2+ is necessary for the proper transfer of the lipid anchor. Cleavage of the terminal tripeptide (AAX) occurs in the endoplasmic reticulum via a specific protease, before the new terminal cysteine is enzymically methylated at the carboxyl group with S-adenosyl methionine as the methyl donor (further increasing the hydrophobicity of the proteolipid). GGT-2 is known to transfer two geranylgeranyl units to the C-terminal double-cysteine motif (CC or CXC) of the 'Rab' family of proteins.

Prenylation of proteins

Prenylation alone tends to target proteins from the cytosol to endomembranes, such as the endoplasmic reticulum and Golgi, a process facilitated by several binding or chaperone proteins, but S-acylation (palmitoylation) can then occur to increase the affinity for membranes before proteins are directed from the endomembranes to the plasma membrane; Ras proteins are transported from the Golgi to the plasma membrane by this means. As the second modification is reversible, it may be part of a control mechanism. After they have been fully processed, these proteolipids have a high affinity for cellular membranes and possess a unique structure at their carboxyl termini, which is a specific recognition motif in some protein-protein interactions. Rho proteins are an exception in that they can be anchored to the plasma membrane, endomembranes or endosomes by geranylgeranylation alone. Although the prenylation reaction is irreversible, some geranylgeranylated Rho proteins can be removed from membranes and in effect de-activated by binding via specific protein-protein interactions; prenylated proteins can also be trafficked between membranes in this way. Binding between prenylated and non-prenylated proteins can serve to increase the activity of the latter, and many examples of this have now been documented.

Protein farnesylation (Ras) and geranylgeranylation (Rho) are critical for Treg cell differentiation, maintenance and function. Prenylated proteins of the Ras family are attached to the cytoplasmic face of the cell membranes, where they transfer the signal from surface receptors to transcription factors for gene expression in the nucleus. In C. elegans, geranylgeranylation of the small G protein RAB-11.1 for interaction with the nuclear hormone receptor NHR-49 is part of a mechanism that enables cells to sense metabolic demand due to lipid depletion and respond by increasing nutrient absorption and lipid metabolism.

Ultimately, degradation of prenylated proteins in animals occurs in the lysosomal compartment of the cell and is catalysed by a prenylcysteine lyase, which is a flavin-containing monooxygenase that converts prenylcysteine to prenyl aldehyde by a novel mechanism.

As many of these proteins are involved in the development of cancer, they are the subject of much pharmaceutical interest, focusing especially on the inhibition of the prenylation reaction with Ras proteins and a farnesyltransferase inhibitor for patients with acute myeloid leukaemia is undergoing clinical trials. Inhibitors of protein farnesyltransferase have been shown to be efficacious in the treatment of protozoal pathogens and other parasitic diseases in animal models, and they appear to be of value in the treatment of viral (e.g., hepatitis D) and fungal infections. Defects in prenylation or its regulation have been implicated in the rare disease progeria, and in cardiovascular disease, neurodegenerative disorders and metabolic diseases.

In plants, protein prenylation is required for plant growth, meristem development and environmental (stress) responses, including the control of abscisic acid and auxin signalling. At least 250 proteins in Arabidopsis have the CAAX sequence and have the potential to be prenylated, although this has been demonstrated experimentally for only a few. It appears that some prenylated proteins do not use the prenyl group to bind to membranes, but this does permit otherwise hydrophilic proteins to operate as peripheral lipid membrane proteins or as a signal for interaction with other proteins. The genome of Arabidopsis encodes two proteases and two methyltransferases, which process prenylated proteins in the endoplasmic reticulum.

5.  O-Acylated Proteins and Peptides

Wnt proteins: In a few proteolipids, serine or threonine residues are acylated such that an O-acyl rather than an S-acyl linkage is formed, and the family of 'Wingless' or Wnt proteins in animals (19 members in humans) falls into this category. These are central mediators of embryonic development and tissue renewal that influence cell proliferation, differentiation and migration, and they need O‑acylation for secretion and activity. They are secreted glycoproteins, which share a conserved sequence of cysteine residues and an N‑terminal signal sequence that targets them for secretion. In all, there is an unusual O‑acyl modification with palmitoleic acid (9‑cis‑hexadecenoic or 9‑16:1), which has been termed a lipokine (i.e., a lipid hormone), at a conserved serine residue (and not with S‑palmitoylation on a conserved cysteine residue as originally reported, although some members of the Wnt family undergo a further S‑acylation with palmitate at a cysteine residue). Following synthesis, Wnt proteins are processed in the endoplasmic reticulum, where the conserved signal sequence is cleaved before glycosylation and fatty acylation.

Desaturation of palmitoyl-CoA to generate a dedicated pool of palmitoleoyl-CoA occurs in the endoplasmic reticulum by the action of stearoyl-CoA desaturase (SCD1), and the subsequent O-acylation uses an O-acyltransferase termed 'porcupine' (PORCN), which is entirely distinct from the S‑acyltransferases (one inhibitor of this enzyme is undergoing clinical trials for treatment of Wnt-driven solid tumours). The specificity of the reaction is due to a structural feature in the enzyme that will only permit the use of the cis-9-monoenoic fatty acid; only those Wnt proteins esterified in this way can separate from the PORCN-Wnt complex. PORCN is a member of the MBOAT family (membrane-bound O-acyl transferase) of multi-pass transmembrane proteins like GOAT and Hhat (see below) that effects fatty acylation of secreted signalling proteins as well as having an involvement in glycerolipid metabolism.

The O-palmitoleoyl residue is essential for intracellular trafficking and operation of Wnt proteins within cells and for maintaining their structural integrity, and it assists their secretion by facilitating movement from the trans Golgi network to the plasma membrane in signalling cells. The transport mechanism involves transfer to the co-chaperone 'Wntless' or 'WLS' (G protein-coupled receptor 177 (GPR177)), a conserved membrane protein that binds to the palmitoleoyl residue of Wnt proteins and facilitates transport by vesicular or non-vesicular means to the plasma membrane and then across it. Serine acylation may be required for extracellular long-range transport of Wnt proteins in lipoprotein particles with protein chaperones or in extracellular vesicles. To initiate signalling, Wnt proteins bind to cell-surface receptors of the 'Frizzled' receptor family, where the palmitoleoyl residue fits neatly within a hydrophobic cleft to which it binds to enable ligand-receptor interactions. Wnt proteins are deactivated by an extracellular carboxylesterase ('Notum'), which removes the palmitoleoyl group.

Wnt proteins are relatively hydrophobic and are stabilized in the aqueous environment of the cell by binding in a hydrophobic space created by specific glypicans, i.e., heparan sulfate proteoglycans that are bound to the outer surface of the plasma membrane by a glycosyl-phosphatidylinositol anchor. These serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.

Formula of ghrelin Ghrelin: Ghrelin is an O-acylated proteolipid, i.e., a circulating 28-amino acid peptide hormone, which is octanoylated (C8) at a serine residue (third amino acid from the N-terminus). It is a hunger-stimulating (anorexigenic) hormone produced in endocrine cells of the human stomach and pancreas that increases caloric intake, decreases energy expenditure and promotes fat deposition. Only the octanoylated protein binds to the single receptor characterized to date, i.e., growth hormone secretagogue receptor GHS‑R1a, which results in increased intracellular Ca++, release of growth hormone and recruitment of multiple proteins to initiate a variety of cell signalling cascades associated with the physiological and behavioural effects of ghrelin. This receptor can stimulate the phospholipase C pathway with accumulation of diacylglycerols and inositol phosphates and thence signalling that leads to the mobilization of intracellular calcium (Ca2+) stores and further downstream events. The picture is further complicated by a finding that GHS-R1a can dimerize and alter the signalling of other G protein-coupled receptors, while the liver-expressed antimicrobial peptide 2 (LEAP2) is an endogenous antagonist for GHS-R1a and is involved in the regulation of ghrelin signalling.

The addition of the octanoyl chain is necessary to induce a conformational change in ghrelin with formation of a hydrophobic core that promotes access to the receptor ligand-binding pocket. Octanoic acid is presumably obtained by β-oxidation of long-chain fatty acids in ghrelin-producing cells, before the ghrelin O‑acyltransferase (GOAT), a member of the MBOAT family of acyltransferases, catalyses octanoylation of pro-ghrelin in the Golgi. This reaction is unique in that ghrelin is the only known octanoylated protein and the only known substrate for this enzyme. After translocation to the endoplasmic reticulum, ghrelin is generated from octanoyl-proghrelin by the action of prohormone convertase 1/3. Mature ghrelin is stored within secretory granules of X/A-like cells, and upon fasting, it is released into the circulation to stimulate appetite. GOAT is now perceived as a target for pharmaceutical intervention in the treatment of the metabolic syndrome. Some circulating esterases can deacylate ghrelin, including the extracellular carboxylesterase ('Notum'), which removes the palmitoleic acid group from Wnt proteins.

Other O-acylated proteins: Aside from ghrelin and the Wnt proteins, relatively few proteins appear to be O-acylated with long-chain fatty acids. One exception is O‑palmitoylation of a serine residue on the protein histone H4 catalysed by the acyl-CoA:lysophosphatidylcholine acyltransferase I (LPCAT1), better known for generating the pulmonary surfactant dipalmitoylphosphatidylcholine from lysophosphatidylcholine. This is presumed to be a mechanism for regulating mRNA synthesis that may lead to changes in global transcription in the cell. O-Palmitoylation of the threonine residue in the C-terminus of the spider venom neurotoxin PLTX-II is believed to regulate toxin potency. In the model nematode C. elegans, serine can be O-acylated with many different fatty acids, including eicosapentaenoic acid.

6.  Hedgehog Proteins - linked Covalently to Cholesterol and Palmitate

Hedgehog (Hh) proteins have a major role in signalling during the differentiation of cells in the development of all embryos from fruit flies to fish to humans, while after embryogenesis, Hh signalling coordinates reparative and regenerative responses in many tissues. They were first found and studied in the insect model Drosophila melanogaster (and named for an anomalous cuticular feature reminiscent of a hedgehog's spines in a mutant), but they are now known to occur in all higher organisms. Vertebrates express three hedgehog family proteins designated 'Sonic' (Shh), 'Indian' (Ihh) and 'Desert' (Dhh) hedgehog, of which Shh has attracted most interest, and they are required for an extensive range of processes from the control of left-right asymmetry of the body to the specification of individual cell types within the brain and to limb development. In endocrine tissues, Hh signalling triggers conversion of cholesterol to progesterone and estradiol. A unique feature is that these proteins contain both palmitic acid and cholesterol in covalent linkage, further confirmation if needed of the vital importance of these occasionally controversial lipids in animal tissues. Proteins that are functionally analogous but structurally distinct are found in nematodes.

Formula of a 'hedgehog' signalling protein

Hedgehog proteolipids are formed post-translationally by attachment of cholesterol via an ester bond to glycine at the C-terminus, a highly conserved region of the protein, while a palmitoyl moiety is attached to a cysteine residue at the N-terminus (N-palmitoylation). The signalling proteins are synthesised initially as ~45 kDa inactive propeptides with two characteristic domains, i.e., an N-terminal 'hedge' domain and a dual function C-terminal autoprocessing and cholesterol recognition domain. After the signalling sequence is removed (1), there is a unique reaction (2) in the endoplasmic reticulum (ER) in which there is an intramolecular attack of an internal cysteine on the carbonyl of an adjacent glycine, resulting in a rearrangement whereby a peptide bond is replaced with a labile thioester bond. This is followed by a transesterification reaction of the thioester bond with the hydroxyl group of a cholesterol moiety (3) facilitated by the processing domain. Cholesterol is thus bound covalently to the C-terminus of the N-terminal signalling domain and simultaneously cleaves this from the processing domain at the cholesteroylation site. Finally, the nascent Hh-N is further modified by the addition of an N-palmitoyl group at Cys-24 (4) at the ER, a reaction mediated by a palmitoyl acyltransferase (Hedgehog acyltransferase or Hhat, another MBOAT family member) to create a highly hydrophobic molecule that is often described as 'Hh-Np for Hh-N-processed' and is transported by a secretory pathway to the plasma membrane.

hedgehog protein maturation

Unlike the other lipid-modified proteins discussed above, but like the GPI-anchored proteins, the lipid moieties are located in the exoplasmic or exterior leaflet of the plasma membrane with the protein component in the extracellular region. The process is regulated both positively and negatively by various oxy-cholesterol derivatives including vitamin D3 and downstream metabolites of 7‑dehydrocholesterol, possibly by competing for binding with sterol-sensing domains (like those in proteins involved in cholesterol homeostasis) in receptor and other proteins, and the interactions between sterol metabolism and hedgehog signalling are increasingly a focus for research.

A hedgehog protein in the outer leaflet of a membraneFollowing biosynthesis in the endoplasmic reticulum and Golgi, there are mechanisms to transport the final proteolipid from the plasma membrane to other cells often for an appreciable distance from the site of synthesis (as much as 30 cell diameters), but much remains to be learned of how this is accomplished. Both lipid components are essential for the proper tissue distribution and efficient operation of the attached proteins, and the N-palmitoyl moiety causes the proteins to form active multimeric complexes, while the cholesterol modification contributes to the partitioning of hedgehog proteins into plasma lipoprotein complexes for long-range transport.

In Drosophila and vertebrates, a protein termed 'Dispatched' (DISP), which contains a sterol-sensing domain, is used for transport through the plasma membrane and is dependent on the trans-membrane sodium (Na+) gradient across the membrane, while cell-surface heparan sulfate proteoglycans, consisting of a glycosylphosphatidyl-inositol (GPI)-linked protein core to which varying numbers of linear glycosaminoglycan chains are attached, may be required. Extracellular extraction from the membrane and subsequent distribution of cholesterol-modified Shh is enhanced by its interaction with the secreted protein SCUBE2 (and analogues) with the cholesterol component essential for this purpose. Cholesterol-modified Shh is also shed from the surface of producing cells in exovesicles or "exosomes” derived from the budding of cellular membranes. In insect models, these proteolipids are transported in the form of lipoprotein complexes (lipophorins) with the lipid moieties in a phospholipid monolayer that surrounds a core of triacylglycerols and cholesterol esters.

The mammalian Shh receptor termed 'PTCH1 or Patched' is a transmembrane protein located on the primary cilium, an antennae-like projection of the plasma membrane from the cytoplasm of the cell. Although the ciliary membrane is contiguous with the plasma membrane, it has a distinctive lipid composition and is relatively enriched in phosphatidylinositol 4-phosphate. PTCH1 contains a sterol-sensing domain and controls the cholesterol composition of the ciliary membrane. When it binds to hedgehog proteins, a second G protein-coupled receptor designated 'SMO or Smoothened' is enabled to enter the primary cilium and initiate down-stream signalling by triggering the expression of target genes through a complex network of post-translational processes and translocations. Cholesterol and oxysterols, including 20S‑hydroxycholesterol, 24S,25‑epoxycholesterol and 25R,26-hydroxy-7-oxocholesterol, can bind to the extracellular cysteine-rich domain of SMO and induce hedgehog signalling. In this process, it has been determined that human SMO is modified by cholesterol through an ester bond between the 3β‑hydroxyl group of the latter and the carboxyl group on the side chain of Asp95 of SMO.

Abnormalities in the expression and/or signalling of the Shh hedgehog proteins have been implicated in developmental (morphological) abnormalities, and these can be fatal to embryos and have been linked to disease states in humans. In adults, aberrant metabolism of Hedgehog proteins, especially of Hhat, promotes tumorigenesis in many different cancers, including those of the pancreas, breast and lung, and inhibition of this protein has therapeutic potential.

7.  Other Proteolipids

N-Acetylation of the primary amine in the ε-position of lysine residues of histone proteins in eukaryotes is a process that leads to neutralization of the position’s positive electrostatic charge in a dynamic mechanism for regulation of chromatin formation and function. The acetylated lysine residues interact with a group of proteins, the so-called "readers”, which contain specific acetyl-lysine binding domains and couple acetylation to various down-stream effects including gene transcription. The reaction is reversible; acetate is added from acetyl-coA by lysine acetyl transferases and removed by lysine deacetylases, while some lysine acetyl transferases can catalyse reaction with other short-chain acyl moieties, including crotonylation, succinylation and propionylation. Although the histone proteins, such as the sirtuins, were the first to be identified, it is now recognized that many other proteins are acetylated in this way in both prokaryotes and eukaryotes. N‑Terminal acetylation of proteins by specific acetyl transferases can occur, but in contrast this is an irreversible reaction, which determines the subcellular localization for certain proteins and modulates protein-protein interactions for normal development of bone, blood vessels and other tissues.

N-Butyrylation of lysine residues of proteins has also been observed in plants and animals, and in the latter has been linked to the onset and progression of several diseases, such as cancer and cardiovascular diseases. In rice, butyrylation is involved in gene expression and thus in growth, development and metabolism.

For practical reasons, the GPI-anchored proteins are discussed elsewhere in these web pages, as are the ceramides and related lipids bound to proteins in skin. In yeasts, a covalent conjugate of phosphatidylethanolamine with a protein designated ‘Atg8’ is involved in the process of autophagy (controlled degradation of cellular components) by promoting the formation of membrane vesicles containing the components to be degraded.

Recommended Reading

An S-palmitoylation database is accessible via Blanc, M., David, F., Abrami, L., Migliozzi, D., Armand, F., Bürgi, J. and van der Goot, F.G. SwissPalm: protein palmitoylation database. F1000Res., 4, 261 (2015);  DOI.

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