PROTEOLIPIDS

STRUCTURE, OCCURRENCE AND BIOLOGY

In 1951, proteins that were soluble in organic solvents such as chloroform-methanol were found in rat brain myelin, although it was another twenty years before it was shown that they contained covalently bound fatty acids and so differed from the plasma lipoproteins. Such lipid-modified proteins are now known to be widespread in nature with a variety of important functions. The term proteolipid is used to define all proteins containing covalently bound lipid moieties, including fatty acids, isoprenoids, cholesterol and glycosylphosphatidylinositol. The last of these ('GPI-anchored proteins') are discussed elsewhere in these web pages. The term ‘lipoprotein’ is also 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.

It is a curious but important fact that only two types of protein with a fatty acid modification have been described , i.e. those with only myristoyl and those with predominantly palmitoyl moieties (both saturated), each with a distinctive type of linkage, amide or thiol ester, respectively. The prenylated lipids contain an isoprenoid group linked via a sulfur atom (thiol ether bond) to the protein.

Formulae of myristoylated,
palmitoylated and prenylated proteins

Modification with lipids occurs after synthesis of the proteins and the effect is to change them from a generally hydrophilic nature to one that is hydrophobic at one end at least, facilitating the interaction with membranes. It is now clear that such modification is important in determining the activities of proteins and in targeting them to specific subcellular membrane domains, including the rafts or 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. receptors, G-proteins, protein tyrosine kinases) and often their substrates are modified by lipids with implications for the relevant signalling events at the cell surface. A further important class of proteolipids contains a linkage to cholesterol in addition to N-palmitoylation.

1. N-Myristoylated Proteins

In the N-myristoylated proteins, myristic acid (14:0) specifically, which is a ubiquitous but usually minor component of cellular lipids, is bound to the amino-terminal glycine residue (of a relatively conserved sequence of the protein) via an amide linkage that is relatively stable to hydrolysis. These constitute a large family of essential eukaryotic and viral proteins with many different functions, and they are located either in the cytosol or in the cytosolic (inner) membrane of cells, or both. The acyl group anchors the protein to the membrane, although simultaneous binding to phospholipids or other membrane constituents is necessary to increase the strength of the interaction.

Scottish thistle In vivo, the acyl group as the CoA ester is attached by the action of a specific transferase to the N-terminal glycine (for which there is an absolute requirement) of the growing peptide as it begins to emerge from the ribosome , i.e. it is a co-translational rather than a post-translational event. Thus, when the initiator methionine residue is removed from the nascent peptide chain, an enzyme myristoyl-CoA:protein N-myristoyltransferase catalyses the formation of the stable amide bond. The functions of these proteolipids are still only poorly understood, although they are certainly of critical physiological importance, especially as participants in cellular signalling processes. They are involved in regulating protein activity perhaps by modifying their conformations, and in targeting otherwise soluble proteins to the membranes and to appropriate receptors. However, a single N-myristoylation is insufficient to target to raft domains specifically. Increased levels of N-myristoylation have been observed in certain cancers, and there have been suggestions that this could be a target for therapeutic intervention. Generally, N-terminal acylation is believed to be an irreversible modification because of the strong chemical nature of amide bonds, but there may be exceptions.

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, in addition to 14:0. Also, myristoylation occurs on the e-amine group of internal lysines in interleukin 1α and tumor necrosis factor alpha. With the latter, different enzymes from those for N-terminal myristoylation are involved. In addition, N-palmitoylated proteins have been found, usually where there is a dual lipid modification. For example, the cholesterol-linked hedgehog proteins (see below) are N-palmitoylated.

Mass spectrometry is currently a key method 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. For example, treatment with 6M HCl or 2M HCl in 83% methanol at 100°C for several hours is required to release the N-acyl group as the free fatty acid or methyl ester, respectively.

2. S-Palmitoylated Proteins

In the S-palmitoylated proteins, palmitic acid (16:0) is linked to one or more (up to four) internal cysteine (or occasionally threonine or serine) residues via labile thioester bonds. The name is something of a misnomer in this instance, as other fatty acids are sometimes present, including 16:1, 18:0 and 18:1. Some N-myristoylated proteins may also be S-palmitoylated internally. The thio-acylated residues can be in many different regions of the proteins, either cytoplasmic or within a predicted trans-membrane domain. In contrast, other lipidation reactions have strict structural requirements within the protein.

The hydrophobic proteolipid protein ('PLP') is the main protein in the myelin of the central nervous system, and was the first of this type to be identified and properly characterized. However, a wide variety of different palmitoylated proteins, with many different functions are now known. For example, fifty different palmitoylated proteins have been identified in the yeast Saccharomyces cerivisiae. 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). Thio-acylation occurs post-translation of the protein, and is catalysed by specific membrane-bound acyltransferases, although there is some evidence for non-enzymatic palmitoylation. Enzymatic mechanisms predominate and protein acyltransferases were identified definitively first from yeasts and subsequently from mammalian cells. A family of such enzymes has now been characterized with a conserved cysteine-rich domain containing a distinctive aspartate-histidine-histidine-cysteine (DHHC) motif, which is required for activity.

protein palmitoylation-depalmitoylation

The protein transacylases are all palmitoylated spontaneously when incubated with palmitoyl-CoA, suggesting that an auto-palmitoylated acyl-enzyme intermediate is involved in the transfer of the palmitoyl moiety to a substrate. However, an alternative suggestion is that the auto-palmitoylation is a regulatory mechanism that facilitates binding of the enzyme to its lipid and protein substrates. There is an absolute requirement for long-chain acyl-CoA esters (16:0, 16:1, 18:0, 18:1) as fatty acyl donors.

As with the myristoylated proteins, palmitoylation is believed to modify protein function partly by modifying their conformations, but mainly in targeting otherwise soluble proteins to specific membranes or to appropriate receptors. The number of bound fatty acyl groups may control the strength of the interaction with membranes. For example, a hydrophobic protein with a single acylation can bind only loosely to membranes and is easily displaced. However, a second or further acylation ensures strong targeting of a protein to the cytoplasmic face of the membrane, and ensures that it is firmly bound to a specific site on the membrane where an appropriate receptor may be located. Palmitoylation of integral membrane proteins may be a regulatory function, increasing their stability by protecting them from degradation by preventing ubiquitylation. In addition, palmitoylation is believed to be an important factor in the process of trafficking proteins between organelles and in directing them to specific membrane compartments. For example, in neurons, palmitoylation targets proteins for transport to nerve terminals and may regulate trafficking at synapses. More generally, palmitoylation facilitates transfer of proteins to lipid rafts, subdomains of the plasma membrane that are enriched in sphingolipids and cholesterol. In this situation, proteolipids can participate in cell signalling events, especially in T-cells of the immune system.

Scottish thistle In addition, palmitoylation is involved in lipoprotein metabolism. Lipoprotein particles containing apolipoprotein B (apoB), such chylomicrons, very-low-density and low-density lipoproteins, are essential for the transport of triacylglycerols and cholesterol esters in plasma. It has been established that palmitoylation of apoB regulates the biogenesis of the nascent lipoprotein particles that contain this apolipoprotein and may regulate the amount available for lipid transport.

In contrast to N-myristoylation and other lipidation reactions, the reverse process of hydrolysis of S-palmitoylated proteins occurs readily, catalysed by thioesterases and in particular by a lysosomal palmitoyl-protein thioesterase. Thus, most proteins of this type undergo cycles of acylation-deacylation, with a half-life that is much shorter than that of the peptide per se. This permits proteins to shuttle between membranes and other cellular compartments, for example between the plasma membrane and Golgi. Indeed synthesis and hydrolysis are regulated dynamically by extracellular stimuli, like phosphorylation, and the level of palmitoylation is determined by a finely tuned balance between the activities of protein acyltransferases and palmitoyl-protein thioesterases. Glycoproteins of viral membrane are exceptions in that they are palmitoylated at or near the cytoplasmic face and then remain palmitoylated.

Although many S-acylated proteins are known to be present in plants, their study lags behind that in animal tissues. However, Ca²⁺ signalling, movement of potassium ions, and stress signalling are amongst the various reported functions.

Analysis by modern mass spectrometric methods permits location of the acyl group to specific amino acids. 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.

3. Prenylated Proteins

Prenylated proteins are formed by attachment of isoprenoid lipid units, farnesyl (C₁₅) or geranylgeranyl (C₂₀), via cysteine thio-ether bonds at or near the carboxyl terminus. Such proteins are ubiquitous in mammalian cells, where they can amount to up to 2% of the total proteins, and they are increasingly being found in plants and microorganisms. Isoprenylation is a stable (non-reversible) modification, which targets specific proteins to membranes and aids protein-protein interactions; it is essential for their functions. However, 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 one or two aliphatic amino acids (A) then to a variable carboxyl-terminal amino acid residue (X). Proteins involved in cellular signalling and trafficking pathways are most involved, and the most important of these are probably the Ras super-family (low-molecular weight G-proteins, or guanosine 5’-triphosphate (GTP) hydrolases), which act as molecular switches for many different signal pathways including those controlling cell proliferation, adhesion, apoptosis and migration, and the integrity of the cytoskeleton.

Scottish thistle Biosynthesis 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 transferase, i.e. protein farnesyltransferase and protein geranylgeranyl transferase (two types), respectively. The enzymes transfer the isoprenoid group to either one or two cysteine residues near the C-terminus. Cleavage of the terminal tri-peptide (AAX) then occurs in the endoplasmic reticulum via a specific protease, and the new terminal cysteine is enzymically methylated at the carboxyl group with S-adenosyl methionine as the methyl donor.

S-Acylation (palmitoylation) of prenylated proteins can occur also, increasing the affinity for membranes and influencing subcellular targeting. As the second modification is reversible, it may function as part of a control mechanism. further subcellular targeting. 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 functions as a specific recognition motif in some protein-protein interactions.

The "Ras" proteins in mammalian cells are farnesylated, while a subfamily of "Rho" proteins are usually geranylgeranylated. As these are involved in the development of cancer, they are the subject of much pharmaceutical interest, focusing especially on the inhibition of the prenylation reaction. In addition, inhibitors of protein farnesyltransferase have been shown to be efficacious in the treatment of protozoal pathogens and other parasitic diseases in animal models. These also appear to be of value in the treatment of viral and fungal infections.

Degradation of prenylated proteins 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.

4. Proteins Linked Covalently to Cholesterol

Cholesterol is found in covalent linkage to specific proteins, known as the "hedgehog" signalling family of which the best studied is the sonic hedgehog (Shh) group. These are formed post-translationally by attachment of cholesterol via an ester bond to glycine in a highly conserved region of the protein(C-terminus)), while a palmitoyl moiety is attached to a cysteine residue at the N-terminus. They were first found and studied in insects, but they are now known to occur in higher organisms, including vertebrates from fish to humans. Proteins that are functionally analogous but structurally distinct are found in nematodes.

Formula of a 'hedgehog' signalling protein

During the biosynthesis of the signalling component, the amino-terminal domain is cleaved from a 45kDa precursor protein at a specific glycine residue by an autocatalytic event, followed by internal proteolysis at a conserved sequence. Cholesterol is then attached to glycine in the carboxy-terminal domain of the new 19 kDa peptide, while the amino-terminal cysteine is palmitoylated, via palmitoyl-CoA and a specific palmitoylacyltransferase, with formation of an amide bond as opposed to a thioester bond as in other proteolipids. Unlike the other lipid-modified proteins discussed above, but like the GPI-anchored proteins, the cholesterol moiety (and the palmitoyl residue) is located in the exoplasmic or exterior leaflet of membranes 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 the interactions between sterol biosynthesis and hedgehog signalling are increasingly a focus for research.

Insertion of hedgeog protein into a lipoprotein membrane The reactions are believed to occur in the endoplasmic reticulum and Golgi, and there must be mechanisms to transport the protein or its active component through the membranes and onwards to other cells, but little is known of how this is accomplished. In insect models, there is evidence that these proteolipids are transported in the form of lipoprotein complexes with the lipid moieties in the phospholipid monolayer that surrounds the core of triacylglycerols and cholesterol esters.

While the cholesterol anchor does not appear to be essential for the function of the proteins, it is required to direct or specify the properties of the signalling activity. The N-terminal palmitate is essential and regulates the strength of Shh signalling. These proteolipids are believed to have a major role in signalling during the differentiation of cells in the development of all embryos from Drosophila to humans. Indeed, they are required for a considerable range of processes, from the control of left-right asymmetry of the body to the specification of individual cell types within the brain and limb development. Aberrant expression and/or signaling of the Shh hedgehog proteins has been implicated in the generation of many human cancers. If further confirmation is needed, this illustrates once more the vital importance of cholesterol in animal tissues.

There are recent suggestions that oxysterols other than cholesterol may link covalently to hedgehog proteins and have distinctive functions.

5. Bacterial Proteolipids

Formula of a bacterial proteolipid All bacteria contain large numbers of proteins (more than 2000 have been identified) with a unique and distinctive post-translational lipid modification that appears to be essential for their efficient function, and even for their pathogenesis via host-pathogen interactions. The lipid components consist of N-acyl- and S-diacylglycerol groups attached to an N-terminal cysteine, i.e. it contains a thio ether bond. As with other proteolipids, the lipid moiety acts as an anchor to hold the protein tightly to a hydrophobic cellular membrane while permitting it to operate in an aqueous environment in such important activities as transport, signalling, adhesion, digestion and growth. They are important constituents of the outer membranes of both Gram-positive and Gram-negative bacteria and like the endotoxins (lipopolysaccharides) of Gram-negative bacteria, they are potent stimulants of the human immune system, eliciting pro-inflammatory immune responses.

In the main secretory pathway, proteins destined to become lipidated have N-terminal signal peptides containing a motif known as a lipobox, which directs them to the lipoprotein biogenesis machinery after transport mainly in an unfolded state. The three fatty acyl groups and the glycerol component responsible for binding to the membrane surface are derived from bacterial phospholipids, especially phosphatidylglycerol. Three enzymes are involved in the biosynthetic pathway. The first attaches the diacylglycerol group from phosphatidylglycerol to the thiol of cysteine, the first amino acid after a signal peptide, in the pro-lipoprotein. A second enzyme then removes the signal peptide, while the third acylates the N-terminal amine group of the modified cysteine with a fatty acid from whatever phospholipid is available.

Note that the terms ‘lipoprotein’, ‘lipopeptide’ and ‘proteolipid’ are used interchangeably for these compounds in the literature. As discussed in the Introduction to this document to avoid confusion, I prefer to reserve the term ‘lipoprotein’ for the non-covalently linked protein-lipid complexes in plasma.

Lipopeptides: A number of bacterial species produce lipopeptides of which the best known are probably the glycopeptidolipids from Mycobacteria and surfactin and related molecules from Bacillus subtilis. These now have their own webpage on this site.

6. Other Proteolipids

N-terminal acetylation of certain membrane proteins targets them for transfer to the Golgi or lysosomes. 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.

The AOCS Lipid Library