Protein myristoylation

Abstract Protein myristoylation refers to the cotranslational addition of a myris-toyl group to an amino-terminal glycine residue of a protein by the enzyme IV-myristoyltransferase (NMT). The myristoylation reaction depends on the availability of the cellular pools of coenzyme A and myristate and their subsequent formation of myristoyl-CoA, the substrate of NMT. In this review, we discuss NMT and myristoyl-CoA binding protein from bovine cardiac muscle which was carried out in our laboratory. 

Fig. 17.4 (A) Inhibition of MCBP by NIP71. (B) Deacylation of protein-myristoyl-CoA complex. For details see Raju et al. (1997).

We examined whether the protein-myristoyl-CoA complex could be inhibited by NIP71. The results indicated that NIP71 did not inhibit the formation of com-plexation between MCBP and myristoyl-CoA (Figure 17.4A). On the other hand,... 

Purified MCBP was incubated with [l-14C]myristoyl-CoA in separate tubes and aliquots were removed at selected times. At time 10 min, cytosolic fraction was added to one tube and buffer was added to the other. Aliquots were removed at selected times up to 120 min. The vial which did not contain added cytosol exhibited the formation of a protein-myristoyl-CoA complex, while the tube which contained added cytosol exhibited deacylation (Figure 17.4B). These results suggest that the cytosolic fraction may contain thioesterases/proteinases which could modulate the acylation reaction in vivo (Raju and Sharma 1997). The absence of acyl-complex formation in the cytosol could be due to the presence of either esterases or pro-teinases. It has been reported that porcine phospholipase A contained thioesterase and deacylase activities (Nocito et al. 1996). 

Harper, D. R., Gilbert, R. L., Blunt, C., and Mcllhinney, R. A. 1993. Inhibition of varicella-zoster virus replication by an inhibitor of protein myristoylation. 1. Gen. Virol. 74 1181-1184. 

In addition to creating recognition motifs to recruit proteins, a few PTM can also increase interaction with other species, such as the lipid bilayer of different cellular membranes. These modifications include the formation of GPI-anchored proteins (20), protein myristoylation on the a-amino group of the N-terminal... 

The classic example of a myristoylated protein is the MARCKS protein (myristoylated alanine-rich C kinase substrate). MARCKS is an actin filament cross-linking protein regulated by PKC and calcium-calmodulin (Aderem el al., 1988 Hartwig et al, 1992). Myristoylation of MARCKS is required for effective binding to the actin network at the plasma membrane (Thelen et al.,... 

Methods to detect and characterize cellular protein myristoylation and palmitoylation are invaluable in cell biology, immunology, and virology. Recently, we developed co-alkynyl fatty acid probes for monitoring myristoylation and palmitoylation of cellular proteins. This article describes a biochemical procedure for metabolic labeling of cells with co-alkynyl fatty acids and click chemistry. 

Methods to monitor the status of protein myristoylation or palmitoylation are invaluable for studying cellular fatty acylation and its role in regulating protein behavior (4). Radioactivity has been the standard method for detecting myristoylation and palmitoylation of cellular proteins. Typically, ( H)- or ( T)-labeled myristic and palmitic acids are added to cells for metabolic incorporation into cellular proteins (5, 6). The signal is developed by... 

Four different types of lipid-anchoring motifs have been found to date. These are amide-linked myristoyl anchors, thioester-linked fatty acyl anchors, thioether-linked prenyl anchors, and amide-linked glycosyl phosphatidylinosi-tol anchors. Each of these anchoring motifs is used by a variety of membrane proteins, but each nonetheless exhibits a characteristic pattern of structural requirements. 

Myristic acid may be linked via an amide bond to the a-amino group of the N-terminal glycine residue of selected proteins (Figure 9.18). The reaction is referred to as A -myristoylation and is catalyzed by myristoyl—CoAtprolein N-myris-toyltransferase, known simply as NMT. A -Myristoyl-anchored proteins include the catalytic subunit of cAMP-dependent protein kinase, the ppSff tyrosine kinase, the phosphatase known as calcineurin B, the a-subunit of G proteins (involved in GTP-dependent transmembrane signaling events), and the gag proteins of certain retroviruses, including the FHV-l virus that causes AIDS. 

A variety of cellular and viral proteins contain fatty acids covalently bound via ester linkages to the side chains of cysteine and sometimes to serine or threonine residues within a polypeptide chain (Figure 9.18). This type of fatty acyl chain linkage has a broader fatty acid specificity than A myristoylation. Myristate, palmitate, stearate, and oleate can all be esterified in this way, with the Cjg and Cjg chain lengths being most commonly found. Proteins anchored to membranes via fatty acyl thioesters include G-protein-coupled receptors, the surface glycoproteins of several viruses, and the transferrin receptor protein. 

This family contains more than 40 members subdivided into five subfamilies [4]. The NCS have been involved in phototransduction and regulation of neurotransmitter release. The NCS have two pairs of EF-hands and, unlike CaM and SI00 proteins, possess a consensus myristoylation sequence at the N-terminal responsible for the targeting of the NCS to the membrane. 

Members of the first group of NCS are recoverin and visinin. Recoverin is a 23 kDa myristoylated protein found under normal conditions only in photoreceptor cells (rods and cones). The main function of recoverin is to bind to and inhibit rhodopsin kinase, thereby prolonging the light response. 

Calcineurin homologous protein (CHP) is ubiquitously expressed and has four EF-hand domains and one putative site of myristoylation. 

Fyn is a nonreceptor tyrosine kinase related to Src that is frequently found in cell junctions. Die protein is N-myristoylated and palmitoylated and thereby becomes associated with caveolae-like membrane microdomains. Fyn can interact with a variety of other signaling molecules and control a diversity of biological processes such as T cell receptor signaling, regulation of brain function, and adhesion mediated signaling. 

Farazi TA, Waksman G, Gordon JT (2001) The biology and enzymology of protein N-myristoylation. J Biol Chem 276 39501-39504... 

Myristoylation is the post-translational addition of the 14-carbon fatty acid myristate to the N-terminal glycine of proteins via an amide link. Myristoylation of proteins helps to anchor them to membranes. 

TRAM is subject to control through phosphorylation by protein kinase C-e. It is phosphorylated on serine 16 which is located close to the myristoylation site which is TRAM cannot signal without this phosphorylation or if the myristoylation site has been mutated. 

Some proteins can be posttranslationally modified by the addition of prenyl groups. Prenyl groups are long-chain, unsaturated hydrocarbons that are intermediates in isoprenoid synthesis. The farnesyl group has 15 carbons, and the geranylgeranyl has 20 carbons. They are attached to a cysteine residue near the end of the protein as a thiol ether (Protein-S-R). Other proteins can have a long-chain fatty acid (C14=myristoyl, C16=palmitoyl) attached to the amino terminus as an amide. These fatty acid modifications can increase the association of proteins with the membrane. 

Although many cellular proteins are potential targets for phosphorylation by PKC, the myristoylated alanine-rich protein kinase C substrate (MARCKS) appears to be a major in vivo substrate. MARCKS is an acidic filamentous actin cross-linking protein that is found in high concentrations at presynaptic junctions and that is directed to... 

C2, protein kinase C domain ENTH, epsin N-terminal homology FERM, band 4.1,exrin, radixin, moesin FYVE, Fabl, YOTB, Vacl, EEA1 MARCKS, myristoylated alanine-rich protein kinase C substrate PH, pleckstrin homology. 

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