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

Abstract

SummaryNH2-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 (Ibl) precursors, also contain covalently linked myristate. To date, the site(s) of acylation of these proteins has not been determined. During an evaluation of Ibl acylation, it was observed that [3H]myristate-labded human monocyte lysates contained a prominent 26-kD myristylated protein, which was identified as the tumor necrosis factor ce (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-e-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-e-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.mong the many modifications of newly synthesized proteins, cotranslational acylation with myristic acid has received considerable attention as an important determinant of protein function and intracellular localization (for review, see reference 1). For most myristylated proteins studied thus far, acyhtion occurs via the formation of an amide bond linking the fatty acid to an NHz-terminal glycine residue after the removal of the initiator methionine. This process has been weU characterized and the enzyme responsible, myristyl CoA: protein N-myristyl transferase (NMT), 1 has been cloned (2). However, in a few cases myristylated proteins have been identified that lack the correctly positioned NHz-terminal glycine strictly required for acylation by NMT. These proteins include the insulin receptor, the/~ Ig heavy chain, and 1 Abbreviation used in this paper: NMT, N-myristyl transferase.the Ibl oc and B precursors (3-5). All of these proteins are myristylated by an undescribed enzymatic mechanism that does not involve acylation on NH2-terminal glycines. One potential alternative mechanism for myristyl acylation would be the myristylation of internal lysine residues, using ...

Reptilian Reovirus Utilizes a Small Type III Protein with an External Myristylated Amino Terminus To Mediate Cell-Cell Fusion

“…Some integral membrane proteins are also myristylated at internal lysine or cysteine residues (2,18,49,50,53). In the case of myristylated integral membrane proteins, stable membrane association derives from the TM domain and the myristic acid serves some undefined function other than protein anchoring in the membrane.…”

Characterization of a polyhistidine‐tagged form of human myristoyl‐CoA:protein N‐myristoyltransferase produced in Escherichia coli

“…From the ratio of the catalytic efficiency (Vma /K,) for the two peptides, p60srcPK-A of 3.36: 1, it is apparent that the former peptide is a better substrate for the enzyme as was observed for the bovine brain enzyme [38,441. The peptide derived from atumour necrosis factor (EEALPKKTGGPQG), which does not contain an N-terminal glycine but has been reported to be myristoylated on the internal lysine residues [45], gave no discernible myristoylation with the purified TrcNMT (data not shown).…”

Acyl Transfer from Membrane Lipids to Peptides Is a Generic Process

“…Lipidation via ester formation at serine or threonine residues occurs for some proteins, including myelin 10 and ion channels. 11 Acylation of internal lysine side 5 chains has been documented for phospholipases, 12,13 cytokines, 14,15 tumour necrosis factor, 15 membrane immunoglobulin heavy chain ( m ), 16 cytochrome C oxidase, 17 adenylate cyclase toxin from B. pertussis, 18 lung surfactant protein C 19 and ocular lens aquaporin, the latter protein being oleoylated site-specifically. 20 Hemolysin from E. coli (HlyA) is acylated on the side chain of two specific lysine residues.…”