Identification of HOIL-1 catalysed ester-linked ubiquitylation sites and their deubiquitylation by DUBs

  • Elisha Honami McCrory

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

The atypical E3 ubiquitin ligase HOIL-1 ubiquitylates serine and threonine residues on proteins forming ester bonds. It regulates innate immunity by ubiquitylating components of the myddosome, but the precise sites of ubiquitylation were unknown. In Chapter 3 of this thesis, I exploited a novel technology called “UbiSite” to identify the serine and threonine residues in the IRAK2, IRAK4 and MyD88 components of myddosomes that undergo HOIL-1catalysed ubiquitylation in macrophages stimulated with R848, an activator of the TLR7/8 heterodimer. IRAK2 became ubiquitylated at Ser136, Thr163 and Ser168 and IRAK4 at Ser175, all located in the Pro-Ser-Thr (PST) domain between the Death Domain and Kinase Domain of these proteins. MyD88 was ubiquitylated at Thr41 in the intermediate Domain between the Death Domain and the Toll-Interleukin-Receptor (TIR) domain. I additionally identified lysine residues in these proteins to which ubiquitin was attached, establishing that the ubiquitin chains that become attached to IRAK2, IRAK4 and MyD88 during TLR7/8 signalling are initiated by both isopeptide and ester bonds.

The UbiSite analysis also revealed that four novel inter-ubiquitin linkages are formed during TLR7/8 signalling in which Thr12, Thr14, Ser20 and Thr22 of one ubiquitin form an ester bond with the C-terminal carboxylate of another ubiquitin molecule. These ester-linked ubiquitin dimers were absent in macrophages from mice expressing an E3 ligase-inactive mutant of HOIL-1. These findings increase from 8 to 12 the number of ubiquitin linkage types known to be formed in cells.

Although humans with HOIL-1 deficiency display abnormal immune responses characterised by a combination of autoinflammation and immunodeficiency, they die in their 20’s from cardiomyopathy and subsequent heart failure, caused by the accumulation and precipitation of unbranched glycogen, termed polyglucosan bodies (PB). PB are also observed in the heart and brains of mice expressing an E3-ligase inactive mutant of HOIL-1, indicating that the HOIL-1 E3 ligase has an essential role in preventing the accumulation and precipitation of PB in cells. In Chapter 4 of this thesis, I found that HOIL-1 monoubiquitylates the α1:4-linked oligosaccharide maltoheptaose directly and established by NMR analysis that the ubiquitin was attached to a C6 hydroxyl group of a glucose molecule in maltoheptaose. Modification occurred on any one of four glucose residues of maltoheptaose (glucosyl residues 2-5). From these results, I propose that HOIL-1 recognises the unbranched glucosaccharides that are formed occasionally by an error of metabolism, and ubiquitylates these molecules, marking them for uptake into lysosomes, where they are degraded by the lysosomal acid a1:4 glucosidase.

The observations made in Chapter 4 stimulated an interest in identifying the deubiquitylases (DUBs) that hydrolyse Ubiquitylated (Ub) maltoheptaose. In Chapter 5, I found that 45 of the 70 DUBs I tested were able to cleave Ubmaltoheptaose, 16 of which deubiquitylated Ub-maltoheptaose more rapidly than the standard isopeptide-linked substrate normally used for their assay. The DUBs that deubiquitylated Ub-maltoheptaose most strongly were MINDY4 (MINDY subfamily), OTUB2 and VCPIP1 (OTU subfamily), UCHL3 (UCH subfamily) and USP30 (USP subfamily), which belong to several structurally unrelated families of DUBs. Similarly, the DUBs that deubiquitylated isopeptide substrates but were unable to deubiquitylate Ub-maltoheptaose also belonged to several different DUB families for example, MINDY 1-3 (MINDY subfamily), and AMSH and AMSH-LP (JAMM subfamily). Thus, specificity for either esterbonds or isopeptide bonds is not confined to particular DUB subfamilies. Only two of six DUBs tested that were active towards Ub-maltoheptaose were also able to deubiquitylate a peptide corresponding to the region surrounding Ser365 of HOIL-1, indicating that the DUBs that deubiquitylate ester bonds in proteins and sugars overlap but are not identical.
Date of Award2024
Original languageEnglish
SupervisorPhilip Cohen (Supervisor) & Yogesh Kulathu (Supervisor)

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