A systematic analysis of human transmembrane E3-RING proteins

Jenn, Robert
(2012) A systematic analysis of human transmembrane E3-RING proteins. Doctor of Philosophy thesis, University of Liverpool.

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The reversible covalent conjugation of the small highly conserved ubiquitin protein modifier to selective substrates plays central roles in countless proteolytic and non-proteolytic cellular functions. Substrate protein ubiquitination is co-ordinated by the sequential activity of three distinct classes of proteins: (i) E1-activating enzymes, (ii) E2-conjugating enzymes, and (iii) E3-protein ligases. Really Interesting New Gene (RING) proteins represent the largest family of E3-proteins comprising over half of predicted human E3-ligases. As such, E3-RING proteins play pivotal roles in controlling both specificity and functionality within the ubiquitin system. E3-RING proteins function as catalytically inactive molecular scaffolds that position Ub~E2 and substrate proteins in close proximity for ubiquitination to occur. Within the active ligase complex, E3-RING proteins and E2 conjugating enzymes are believed to select protein substrate(s) and the form of conjugated ubiquitin upon them, respectively. Whilst E3-RING/E2 partners have been investigated in recent HTP screen approaches, a key area of data paucity exists for integral membrane E3-RING (TM-E3-RING) proteins. As such, high throughput yeast-two-hybrid assays were performed for the entire complement of TM-E3-RING proteins and E2-conjugating enzymes. A broad subset of TM-E3-RING/E2 positive and negative Y2H interactions was re-tested in secondary luciferase protein complementation assays (PCAs), which increased confidence in Y2H-derived interactions and extended network coverage. Data from these studies was collated with previously published binary TM-E3-RING/E2 interaction data to provide a high-confidence TM-E3-RING/E2 network consisting of 312 unique binary interactions. In vitro auto-ubiquitination assays were employed to assign functional activity to TM-E3-RING/E2 protein pairs, revealing high verification rates for both positive and negative Y2H or PCA binary interaction data. Furthermore, novel trends in the generation of different forms of ubiquitin modifications were identified between selective TM-E3-RING/E2 pairs. Finally, Y2H screens were also performed to identify TM-E3-RING dimerization events, which represent an emerging theme in ubiquitin system regulation. In total 71 TM-E3-RING/TM-E3-RING interactions were reported demonstrating high incidence of these binding events. Novel data was combined with known interactions to generate a TM-E3-RING network containing >500 binary interactions, encompassing both components of the core ubiquitin cascade and non-ubiquitome proteins. This TM-E3-RINGcentric network provides a valuable tool for the investigation of specificity and regulation of TM-E3-RING proteins and specific ubiquitin cascades.

Item Type: Thesis (Doctor of Philosophy)
Uncontrolled Keywords: E3-RING, transmembrane, Ubiquitin, Y2H, yeast two-hybrid
Subjects: ?? QP ??
Depositing User: Symplectic Admin
Date Deposited: 06 Aug 2012 10:14
Last Modified: 23 Jan 2024 12:44
DOI: 10.17638/00005913
URI: https://livrepository.liverpool.ac.uk/id/eprint/5913