S-acylated proteins

S-acylated proteins include many GTP-binding regulatory proteins (G proteins), including most a subunits of heterotrimeric G-proteins and also many members of the Ras superfamily of monomeric G proteins, a number of G protein-coupled receptors, several nonreceptor tyrosine kinases, and a number of other signaling molecules, -acylation is posttranslational and reversible, a property that allows the cell to control... 

Bone proteins S-acylated proteins Pycnodysostosis Cathepsin K lq21... 

Small cysteine-containing peptides similar to sequences of S -acylated proteins were generally synthesized in solution. The... 

The condition is caused by deficiencies of palmitoyl protein thioesterase 1 (PPTl) (or tripeptidyl peptidase 1 (TPPl) and possibly other enzymes resulting in the same clinical presentation of NCL (for Aeural Ceroid Lipofuscinose)). PPTl cleaves long-chain fatty acids from S-acylated proteins within the lyso-some (Lu et al., 1996). How the loss of this activity causes the death of central nervous system neurons is not known. 

Thioacylated proteins contain fatty acids in thioester linkage to cysteine residues [7-9] (Fig. lA). Protein thioacylation is frequently referred to as palmitoylation, although fatty acids other than palmitate are found on thioacylated proteins. Membrane proteins as well as hydrophilic proteins are thioacylated, the latter, in many cases, acquiring the modification when they become associated with a membrane compartment as a result of initial N-myristoylation or prenylation. Examples include G-protein coupled receptors, the transferrin receptor, the cation-dependent mannose-6-phosphate receptor, and hydrophilic proteins such as members of the Src family of protein tyrosine kinases (e.g., p59h " and p56 ) as well as H-Ras, N-Ras, and the synaptic vesicle protein SNAP-25. The yeast palmitoyl proteome, i.e., the collection of all S-acylated proteins in yeast, was recently defined via a comprehensive proteomics approach (A.F. Roth, 2006). It consists of 50 proteins including... 

These findings led to the conclusion that the regulation of membrane anchored proteins has to be achieved by mechanisms other than spontaneous dissociation. In principal, binding to an escort protein or de-S-acylation may induce dissociation of the lipoproteins out of the membrane structure. 

It is noteworthy that there is another limiting factor in the choice of amino acid types at the junction sites which affect the enzymatic process of the intein. For example, in the case of SceVMA (also called PI-Seel) from the IMPACT system, proline, cysteine, asparagine, aspartic acid, and arginine cannot be at the C-terminus of the N-terminal target protein just before the intein sequence. The presence of these residues at this position would either slow down the N-S acyl shift dramatically or lead to immediate hydrolysis of the product from the N-S acyl shift [66]. The compatibility of amino acid types at the proximal sites depends on the specific inteins and needs to be carefully considered during the design of the required expression vectors. The specific amino acid requirements at a particular splicing site depends on the specific intein used and is thus a crucial point in this approach. 

The acylated peptides (Myr)GCX-Bimane 31 a-e (X = G, L, R, T, V), which are found in certain nonreceptor tyrosine kinases and ct-subunits of several heterotrimeric G-proteins, were synthesized in solution using common solution-phase peptide synthesis with X-myristoylglycine as a building block. These model peptides were used for acylation studies with palmitoyl-CoA in phospholipid vesicles at physiological pH. For such uncatalyzed spontaneous reactions only a modest molar excess of acyl donor species (2.5 1) was necessary. Unprotected side chains of threonine or serine are not interfering with this S-acylation (Scheme 14). 

N-Myristoylation is achieved by the covalent attachment of the 14-carbon saturated myristic acid (C14 0) to the N-terminal glycine residue of various proteins with formation of an irreversible amide bond (Table l). 10 This process is cotranslational and is catalyzed by a monomeric enzyme called jV-myri s toy 11ransferase. 24 Several proteins of diverse families, including tyrosine kinases of the Src family, the alanine-rich C kinase substrate (MARKS), the HIV Nef phosphoprotein, and the a-subunit of heterotrimeric G protein, carry a myr-istoylated N-terminal glycine residue which in some cases is in close proximity to a site that can be S-acylated with a fatty acid. Functional studies of these proteins have shown an important structural role for the myristoyl chain not only in terms of enhanced membrane affinity of the proteins, but also of stabilization of their three-dimensional structure in the cytosolic form. Once exposed, the myristoyl chain promotes membrane association of the protein. 5 The myristoyl moiety however, is not sufficiently hydrophobic to anchor the protein to the membrane permanently, 25,26 and in vivo this interaction is further modulated by a variety of switches that operate through covalent or noncovalent modifications of the protein. 4,5,27 In MARKS, for example, multiple phosphorylation of a positively charged domain moves the protein back to the cytosolic compartment due to the mutated electrostatic properties of the protein, a so-called myristoyl-electrostatic switch. 28 ... 

As discussed for N-myristoylation and S-prenylation, even S-acylation of proteins with a fatty acid which in the vast majority of cases is the C16 0 palmitic acid, plays a fundamental role in the cellular signal-transduction process (Table l). 2-5 14 While N-myristoylation and S-prenylation are permanent protein modifications due to the amide- and sulfide-type linkage, the thioester bond between palmitic acid and the peptide chain is rather labile and palmi-toylation is referred to as a dynamic modification. 64 This reversibility plays a crucial role in the modulation of protein functions since the presence or absence of a palmitoyl chain can determine the membrane localization of the protein and can also be used to regulate the interactions of these proteins with other proteins. Furthermore, a unique consensus sequence for protein palmitoylation has not been found, in contrast to the strict consensus sequences required for N-myristoylation and S-prenylation. Palmitoylation can occur at N- or C-terminal parts of the polypeptide chain depending on the protein family and often coexists with other types of lipidation (see Section 6.4.1.4). Given the diversity of protein sequences... 

Van Lanen SG, Dorrestein PC, Christenson SD, Liu W, Ju J, Kelleher NL, Shen B (2005) Biosynthesis of the (3-Amino Acid Moiety of the Enediyne Antitumor Antibiotic C-1027 Featuring (3-Amino Acyl-S-carrier Protein Intermediates. J Am Chem Soc 127 11594... 

Fig. 2. Scheme of protein splieing. Cleavage pathway proposed for intein that possesses a cysteine residue in eaeh spliee junetion. In the initial step a linear thioester intermediate is formed by an N-S acyl rearrangement at Cysi (N-terminal amino acid of the intein). Next, traw -thioesterification that involves nucleophilic attack of the side-... 

Figure 4 Mechanism of trans-protein splicing, (a) Initial association of the intein halves to form a functional intein. (b) Activation of the N-terminal splice-junction via an N-S acyl shift, (c) Formation of a branched intermediate upon transthioesterification. (d) Branch resolution and intein release by succinimide formation. Spontaneous S-N acyl rearrangement yields the processed product with a native peptide backbone.

Lu J.Y., Verkruyse L.A., Hofmann S.L., Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase. Proceedings of the National Academy of Sciences of the United States of America 93 (1996) 10046-10050. 

A family of protein acyltransferases (PATs) is responsible for S-acylation of proteins in cells (S. Lobo, 2002 A. Roth, 2002) [8]. Members of this family are characterized by the presence of a cysteine-rich domain containing a DHHC (Asp-His-His-Cys) motif. PATs are polytopic membrane proteins with the putative catalytic DHHC motif localized to a cytoplasmic loop between transmembrane spans. Some PATs function alone whereas others, such as the yeast Ras PAT Erf2, require a cytoplasmic protein, Erf4, for activity. It is likely that particular classes of substrate have a dedicated PAT that accounts for most, if not all, of their S-acylation. For example, Swflp modifies SNARE proteins and other monotopic membrane proteins with a juxtamembrane cysteine residue. The yeast vacuolar protein Vac8 is mainly S-acylated by the vacuolar DHHC protein Pfa3 (J.E. Smotrys,... 

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