Myristyl acylation of the tumor necrosis factor α precursor on specific lysine residues

Article (PDF Available)inJournal of Experimental Medicine 176(4):1053-62 · November 1992with14 Reads
Source: PubMed
Abstract
NH2-terminal glycine myristyl acylation is a cotranslational modification that affects both protein localization and function. However, several proteins that lack NH2-terminal glycine residues, including the interleukin 1 (IL-1) precursors, also contain covalently linked myristate. To date, the site(s) of acylation of these proteins has not been determined. During an evaluation of IL-1 acylation, it was observed that [3H]myristate-labeled human monocyte lysates contained a prominent 26-kD myristylated protein, which was identified as the tumor necrosis factor alpha (TNF) precursor protein on the basis of specific immune precipitation. Radioimmunoprecipitates from the supernates of labeled monocytes indicated that the processed or mature 17-kD form of TNF does not contain myristate, suggesting that the site of acylation occurs within the 76-amino acid propiece of the precursor molecule. As the TNF precursor does not contain an NH2-terminal glycine, we hypothesized that myristyl acylation occurs on the N-epsilon-NH2 groups of lysine, of which two are present in the propiece (K19K20). Synthetic peptides were designed to include all seven lysine residues present within the entire 26-kD TNF precursor, and used in an in vitro myristyl acylation assay containing peptide, myristyl-CoA, and monocyte lysate as a source of enzyme. Analysis of reaction products by reverse phase high performance liquid chromatography and gas phase sequencing demonstrated the exclusive myristyl acylation of K19 and K20, consistent with the presence in monocytes of a specific lysyl N-epsilon-NH2-myristyl transferase activity. The acylated lysine residues are located immediately downstream from a hydrophobic, probable membrane-spanning segment of the propiece. Specific myristyl acylation of the TNF propiece may facilitate membrane insertion or anchoring of this critical inflammatory mediator.

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Available from: David H Lovett, Aug 05, 2014
    • "Since O-fatty-acylated proteins are readily labeled with chemical reporters, such as az-15 [49] and alk-16 [42] , these reagents combined with selective ester cleavage methods and proteomic analysis could reveal novel O-fatty-acylated proteins. N e -fatty-acylation: Recently, the discovery of sirtuin-mediated deacylation of e-N-Lys fatty-acylation (Figure 1e) has suggested more prevalent roles for this modification beyond a few cytokines such as TNF-a and Interleukin- 1-a [50,51] . For example, SIRT6, a sirtuin-family deacylase , was shown to hydrolyze specific long chain fatty acids, notably myristate, present on lysine side-chains in mammalian cells using alk-14 metabolic labeling and in vitro biochemical assays [15 ] . "
    [Show abstract] [Hide abstract] ABSTRACT: Protein fatty-acylation in eukaryotes has been associated with many fundamental biological processes. However, the diversity, abundance and regulatory mechanisms of protein fatty-acylation in vivo remain to be explored. Herein, we review the proteomic analysis of fatty-acylated proteins, with a focus on N-myristoylation and S-palmitoylation. We then highlight major challenges and emerging methods for direct site identification, quantitation, and lipid structure characterization to understand the functions and regulatory mechanisms of fatty-acylated proteins in physiology and disease.
    Article · Feb 2016
    • "organization in membranes. The control of ion channel trafficking, from synthesis in the ER through modifica­ tion in the Golgi apparatus to subsequent delivery to the appropriate cellular membrane compartment, is a and oleic acid (oleoylation) as well as myristate via amide linkages on lysine residues (Stevenson et al., 1992; Linder and Deschenes, 2007; Hannoush and Sun, 2010; Schey et al., 2010). These modifications can be discriminated from S­acylation by their insensitivity to hydroxylamine cleavage (at neutral pH) compared with the S­acylation thioester linkage. "
    [Show abstract] [Hide abstract] ABSTRACT: Protein S-acylation, the reversible covalent fatty-acid modification of cysteine residues, has emerged as a dynamic posttranslational modification (PTM) that controls the diversity, life cycle, and physiological function of numerous ligand- and voltage-gated ion channels. S-acylation is enzymatically mediated by a diverse family of acyltransferases (zDHHCs) and is reversed by acylthioesterases. However, for most ion channels, the dynamics and subcellular localization at which S-acylation and deacylation cycles occur are not known. S-acylation can control the two fundamental determinants of ion channel function: (1) the number of channels resident in a membrane and (2) the activity of the channel at the membrane. It controls the former by regulating channel trafficking and the latter by controlling channel kinetics and modulation by other PTMs. Ion channel function may be modulated by S-acylation of both pore-forming and regulatory subunits as well as through control of adapter, signaling, and scaffolding proteins in ion channel complexes. Importantly, cross-talk of S-acylation with other PTMs of both cysteine residues by themselves and neighboring sites of phosphorylation is an emerging concept in the control of ion channel physiology. In this review, I discuss the fundamentals of protein S-acylation and the tools available to investigate ion channel S-acylation. The mechanisms and role of S-acylation in controlling diverse stages of the ion channel life cycle and its effect on ion channel function are highlighted. Finally, I discuss future goals and challenges for the field to understand both the mechanistic basis for S-acylation control of ion channels and the functional consequence and implications for understanding the physiological function of ion channel S-acylation in health and disease.
    Article · May 2014
    • "Tumour necrosis factor-a (TNF-a) is a pleiotropic pro-inflammatory cytokine synthesized as a 26-kDa precursor localized as a transmembrane molecule, which is cleaved by membranebound metalloproteinases, mainly the TNF-a converting enzyme (TACE) to yield the soluble form (Black et al. 1997, Moss et al. 1997). Transmembrane TNF-a is myristoylated in human monocytes on Lys-58 and Lys-57, located within the leader sequence of TNF-a (Stevenson et al. 1992). The functional significance of these modifications has been proposed to regulate TNF-a shedding and therefore its proinflammatory activity in atherogenesis (Utsumi et al. 2001, Canault et al. 2004). "
    [Show abstract] [Hide abstract] ABSTRACT: Lipid-modified proteins are classified based on the identity of the attached lipid, a post- or co-translational modification required for their biological function. At least five different lipid modifications of cysteines, glycines and other residues on the COOH- and NH(2)-terminal domains have been described. Cysteine residues may be modified by the addition of a 16-carbon saturated fatty acyl group by a labile thioester bond (palmitoylation) or by prenylation processes that catalyze the formation of thioether bond with mevalonate derived isoprenoids, farnesol and geranylgeraniol. The NH(2)-terminal glycine residues may undergo a quite distinct process involving the formation of an amide bond with a 14-carbon saturated acyl group (myristoylation), while glycine residues in the COOH-terminal may be covalently attached with a cholesterol moiety by an ester bond. Finally, cell surface proteins can be anchored to the membrane through the addition of glycosylphosphatidylinositol moiety. Several lines of evidence suggest that lipid-modified proteins are directly involved in different steps of the development of lesions of atherosclerosis, from leukocyte recruitment to plaque rupture, and their expression or lipid modification are likely altered during atherogenesis. This review will briefly summarize the different enzymatic pathways of lipid modification and propose a series of lipid-modified proteins that can be used as biomarkers for cardiovascular disease.
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