Thioester

About: Thioester is a research topic. Over the lifetime, 1776 publications have been published within this topic receiving 42869 citations.

Protein ubiquitination involving an E1–E2–E3 enzyme ubiquitin thioester cascade

Abstract: Ubiquitination of proteins involves the concerted action of the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes and E3 ubiquitin-protein ligases. It has been proposed that E3s function as 'docking proteins', specifically binding substrate proteins and specific E2s, and that ubiquitin is then transferred directly from E2s to substrates. We show here that formation of a ubiquitin thioester on E6-AP, an E3 involved in the human papillomavirus E6-induced ubiquitination of p53 (refs 6-10), is an intermediate step in E6-AP-dependent ubiquitination. The order of ubiquitin transfer is from E1 to E2, from E2 to E6-AP, and finally from E6-AP to a substrate. This cascade of ubiquitin thioester complexes suggests that E3s have a defined enzymatic activity and do not function simply as docking proteins. The cysteine residue of E6-AP responsible for ubiquitin thioester formation was mapped to a region that is highly conserved among several proteins of unknown function, suggesting that these proteins share the ability to form thioesters with ubiquitin.

Formation of the initial C3 convertase of the alternative complement pathway. Acquisition of C3b-like activities by spontaneous hydrolysis of the putative thioester in native C3.

Abstract: Activation of the alternative pathway of complement commences with the formation of an initial fluid-phase C3 convertase. Treatment of C3 with the nucleophilic reagent methylamine has previously been shown to result in the cleavage of an intramolecular thioester bond and to induce C3b-like properties, including the ability to form a fluid-phase C3 convertase. This report examines the hypothesis that spontaneous hydrolysis of the thioester generates a derivative of C3 that is responsible for the formation of the initial C3 convertase of the alternative pathway. The rate of spontaneous decay of C3 hemolytic activity in buffer was found to be between 0.2 and 0.4%/h. In the presence of other alternative pathway proteins, the rate of inactivation was 1%/h. The rate of spontaneous inactivation was greatly accelerated by low concentrations of chaotrophic agents such as KSCN or guanidine. Liberation of a sulfhydryl group, not present in native C3, correlated with loss of hemolytic activity, indicating that exposure to chaotropic agents resulted in thioester hydrolysis. Unlike native C3, C3 bearing a single reactive sulfhydryl group was capable of generating fluid-phase C3 convertase with Factors B, D, and P and was cleaved by Factor I (C3b inactivator) in the presence of Factor H (beta 1H). The fragmentation patterns indicated that the C3a domain was covalently associated with the functionally C3b-like C3. Organomercurial agarose was employed for the rapid removal of sulfhydryl-bearing, hemolytically inactive forms of C3 and C3b from native hemolytically active C3.

An Efficient Fmoc-SPPS Approach for the Generation of Thioester Peptide Precursors for Use in Native Chemical Ligation

Abstract: The straightforward C-terminal modification of peptides assembled on a solid support remains a significant challenge in peptide and protein chemistry. In particular, C-terminal thioester peptides are important intermediates for the generation of active esters, amides and hydrazides[1,2] and are an essential component of many synthetic strategies for protein synthesis.[3] Currently, the most effective approach for the synthesis of peptidyl thioesters is the in situ neutralization protocol for Boc solid phase peptide synthesis (Boc-SPPS)[4] using thioester linkers.[2,5] However, many laboratories use Fmoc-SPPS exclusively and such protocols are favored when synthesizing glyco- and phosphopeptides. The thioester linkers used for Boc-SPPS have limited utility for Fmoc-SPPS due to the requirement for repeated Fmoc removal under basic conditions. Considerable effort has been applied to address this challenge[6] including optimized Fmoc deprotection cocktails,[7] thiol labile safety catch linkers,[8] activation of protected peptides in solution,[9] and recently thioesters have been generated using O to S[10] or N to S[11] acyl transfer. Despite these notable advances, the synthesis of thioester peptides by Fmoc-SPPS remains significantly more challenging than the synthesis of the corresponding acid or amide peptide.