Protein acyltransferases

The three CAAX-signaled modifications increase the overall hydrophobicity of the carboxyl terminus of Ras and are necessary to promote Ras membrane association. However, these modifications alone are not sufficient to direct full plasma membrane association and signaling activity. At least two additional motifs exist at the carboxyl termini of Ras proteins, which serve to facilitate plasma membrane association and direct Ras proteins to discrete membrane subdomains. These motifs function as secondary signals and are composed either of a stretch of basic amino acids (K-Ras4B) or of cysteine(s) that are pahni-toylated (H-Ras, N-Ras, K-Ras4A) and positioned immediately upstream of the CAAX motif (28, 30) (see Fig. 3). The addition of a palmitate fatty acid is catalyzed by a protein acyltransferase, which forms a reversible thioester bond between the cysteine and the pahnitoyl group (31, 32). 

Protein acyltransferases (PATs) and thioacyl protein thioesterases (APTs). 45... 

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,... 

DHHC-CRD Asp-His-His-Cys cysteine-rich domain-containing protein acyltransferases... 

Althongh the isoprenoid moiety is essential for localization, it is nsnally not enongh for ensuring stable membrane association. In the case of the K-Ras isoform, this is achieved by a polybasic seqnence formed by eight lysines, which specifically interact with the acidic phospholipids present at the membrane. In some other cases, snch as the N-Ras or H-Ras isoforms, a second lipidation motif is necessary, and these proteins are subsequently palmi-toylated at cysteine residnes located near the prenylated cysteine. The palmitoylation of cysteines is a reversible modification catalyzed by protein acyltransferases (PATs) and by acyl protein thioes-terases (APTs) (Fig. 1) [9]. 

FIGURE 25.20 Triacylglycerols are formed primarily by the action of acyltransferases on mono- and diacylglycerol. Acyltransferase in E. coli is an integral membrane protein (83 kD) and can utilize either fatty acyl-CoAs or acylated acyl carrier proteins as substrates. It shows a particular preference for palmitoyl groups. Eukaryotic acyltransferases nse only fatty acyl-CoA molecnles as substrates. 

High-density lipoproteins (HDL) have much longer life spans in the body (5 to 6 days) than other lipoproteins. Newly formed HDL contains virtually no cholesterol ester. However, over time, cholesterol esters are accumulated through the action of lecithin cholesterol acyltransferase (LCAT), a 59-kD glycoprotein associated with HDLs. Another associated protein, cholesterol ester transfer protein, transfers some of these esters to VLDL and LDL. Alternatively, HDLs function to return cholesterol and cholesterol esters to the liver. This latter process apparently explains the correlation between high HDL levels and reduced risk of cardiovascular disease. (High LDL levels, on the other hand, are correlated with an increased risk of coronary artery and cardiovascular disease.)... 

Apohpoproteins carry out several roles (1) they can form part of the stmcture of the hpoprotein, eg, apo B (2) they are enzyme cofactors, eg, C-11 for lipoprotein hpase, A-1 for lecithinicholesterol acyltransferase, or enzyme inhibitors, eg, apo A-11 and apo C-111 for lipoprotein hpase, apo C-1 for cholesteryl ester transfer protein and (3) they act as hgands for interaction with lipopro-... 

Figure 26-5. Factors affecting cholesterol balance at the cellular level. Reverse cholesterol transport may be initiated by pre 3 HDL binding to the ABC-1 transporter protein via apo A-l. Cholesterol is then moved out of the cell via the transporter, lipidating the HDL, and the larger particles then dissociate from the ABC-1 molecule. (C, cholesterol CE, cholesteryl ester PL, phospholipid ACAT, acyl-CoA cholesterol acyltransferase LCAT, lecithinicholesterol acyltransferase A-l, apolipoprotein A-l LDL, low-density lipoprotein VLDL, very low density lipoprotein.) LDL and HDL are not shown to scale.

Figure 11.2 Biosynthesis of the nine-membered enediynes. Members of this family share a common biosynthetic pathway for the enediyne core intermediate. Domains are shown in circles with abbreviations (KS, ketosynthase AT, acyltransferase KR, ketoreductase DH, dehydratase TE, thioesterase ACP, acyl carrier protein PPT, phosphopantetheine transferase)...

It has recently been demonstrated (191) that the nature and location of lipid A primary fatty acids is determined by the specificity of the enzymes UDP-GlcpNAc-G-acyltransferase and UDP-3-6>-[(i )-hydroxyacyl]-GlcpN-N-acyltransferase for acyl - acyl carrier protein (acyl ACP). The analysis of the acyl ACP specificity of these O- and A-acyltransferases should, therefore, constitute a biochemical approach for elucidation of the location of primary fatty acids in lipid A (191). 

In order to analyze the effect that conformational restriction has on the antibiotic enzymatic inactivation, three different enzymes were chosen as model systems Staphylococcus aureus ANT(4 ), Mycobacterium tuberculosis AAC(2 ) and Enterococcus faecalis APH(3 ). These proteins are representative of the three main families of enzymes that modify aminoglycosides adenyltrans-ferases, acyltransferases and phosphotransferases. In addition, there is high resolution X-ray structural information available for the three enzymes in complex with several antibiotics. 

LCAT, lecithin cholesterol acyltransferase CETP, cholesterol ester transfer protein SR-61, scavenger receptor-BI... 

ACAT Acyl-CoA cholesterol acyltransferase cAPK Protein kinase A (or cyclic AMP-... 

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