A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase.

Abstract

E6-AP is a 100-kDa cellular protein that interacts with the E6 protein of the cancer-associated human papillomavirus types 16 and 18. The E6/E6-AP complex binds to and targets the p53 tumor-suppressor protein for ubiquitinmediated proteolysis. E6-AP is an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. The amino acid sequence of E6-AP shows similarity to a number of protein sequences over an -350-aa region corresponding to the carboxyl termini of both E6-AP and the E6-AP-related proteins. Of particular note is a conserved cysteine residue within the last 32-34 aa, which in E6-AP is likely to be the site of ubiquitin thioester formation. Two of the E6-AP-related proteins, a rat 100-kDa protein and a yeast 95-kDa protein (RSP5), both of previously unknown function, are shown here to form thioesters with ubiquitin. Mutation of the conserved cysteine residue of these proteins destroys their ability to accept ubiquitin. These data strongly suggest that the rat 100-kDa protein and RSP5, as well as the other E6-AP-related proteins, belong to a class of functionally related E3 ubiquitin-protein ligases, defined by a domain homologous to the E6-AP carboxyl terminus (hect domain).The hallmark of the ubiquitin-mediated proteolytic pathway is the covalent attachment of the 76-aa ubiquitin polypeptide to target proteins, through isopeptide bond formation between the carboxyl terminus of ubiquitin and the s-amino group of one or more lysine residues on the protein substrate (reviewed in refs. 1 and 2). Additional ubiquitin moieties can be ligated via lysine residues of ubiquitin itself, resulting in the formation of multiubiquitinated proteins, which are then recognized and degraded by the 26S protease complex. While much of the biochemistry of the ubiquitin proteolysis system has been elucidated, a basic question has remained largely unanswered: how are proteins specifically recognized and targeted for ubiquitination?Protein ubiquitination involves three classes of enzymes. Ubiquitin is activated by the El ubiquitin-activating enzyme in an ATP-dependent reaction, resulting in thioester formation between a specific cysteine of the enzyme and the carboxyl terminus of ubiquitin. The activated ubiquitin is transferred to a cysteine residue of one of a number of low molecular weight E2 ubiquitin-conjugating enzymes. The E2 proteins have generally been thought to catalyze the final ubiquitination of the substrate protein, often in conjunction with a third group of proteins, the E3 ubiquitin-protein ligases. E3 activities have been proposed to play a major role in defining the substrate specificity of the ubiqulitin system, perhaps through direct binding of substrates. In the yeast Saccharomyces cerevisiae at least 12 different E2 genes have been identified (1). Only two known E3 genes have been cloned, yeast UBR1 (3) and the human E6-AP gene (4, 5).E6-AP was discovered in the course of c...

Mathematical modeling of noise and discovery of genetic expression classes in gliomas

“…MDPI, a DNA-binding protein, inhibits the rapid growth of E-Coli and mycobacterium smegmatis, supporting the idea that it may function as a tumor suppressor gene (Matsumoto et al, 2000). In addition, RSP5 interacts with E6-AP ubiquitin -protein ligase that binds and targets the p53 tumor-suppressor protein for ubiquitin-mediated proteolysis (Huibregtse et al, 1995). Clone 141698, a member of class II, is 41% homologous to human oxysterol-binding protein (OXYB).…”

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

“…E3 ubiquitin ligases are classed into three sub‐families according to their catalytic mechanism: HECT and RBR‐type ligases form a thioester intermediate with ubiquitin before its final transfer onto the substrate, though each sub‐family uses different structural features to perform this function (Huibregtse et al , 1995; Wenzel et al , 2011; Smit & Sixma, 2014; Spratt et al , 2014). In contrast, RING E3 ligases act as adaptors to bring the ubiquitin‐loaded E2 together with the substrate and promote ubiquitin transfer without directly participating in the reaction (Berndsen & Wolberger, 2014; Metzger et al , 2014).…”

The HECT E3 ubiquitin ligase Rsp5 is important for ubiquitin homeostasis in yeast

“…The Rsp5 protein of the yeast Saccharomyces cerevisiae is a member of the HECT (homology to E6-AP carboxyl terminus) E3 family of ubiquitin ligases [1]. It is an essential protein under standard growth conditions.…”