Infection and colonisation by microbes is a constant threat to humans and effective, yet balanced, innate and adaptive immune responses to infection is critical for human health. Protein modification by ubiquitin, interferon stimulated gene 15 (ISG15), a di-ubiquitin homologue, and phosphorylation control the signalling processes that define immune responses. Accordingly, genetic mutations and environmental factors that deregulate ubiquitin (Ub) / ISG15 signalling can lead to detrimental and potentially fatal immune-related disorders.
Ub molecules are assembled into chains by E3 Ub ligases (E3) via either of seven lysine (K) residues or the N-terminal methionine (M1) in Ub, which allows for generation of eight different homotypic chains and an almost indefinite number of mixed and branched chain types. These different chain types, underpin the pleiotropic cellular functions of Ub chains in immune signalling where at least three different Ub chain types coordinate receptor signalling responses. Protein modifications via ISG15 are less well understood.
We have pioneered proteomic approaches to study the function and regulation of individual Ub chain types in cellular signalling, including the first proteomic analysis of Ub chains generated by innate immune receptor stimulation. Nonetheless, our knowledge about the linkage-composition and stoichiometry of Ub landscapes generated by innate immune receptors (or other cell signalling processes) remains limited to a few of the eight possible Ub-linkages (Figure 1).
The aim of this project is to develop novel methodologies to facilitate mass spectrometry-based analysis of the composition, regulation and function of the complex Ub landscapes that control immune signalling. This will then be extended to study the cellular ISG15ylation profile including the characterisation of novel ISG15ylases that may participate in these signalling cascades.
The project will involve a range of state-of-the-art methods, including advance mass spectrometry, biochemical and molecular biology, cell biology, CRISPR-based genome editing of cultured cells.
Project reference number: 875
|Professor Mads Gyrd-Hansen||Oxford Ludwig Institute||Oxford University, Old Road Campus Research Building||GBRemail@example.com|
|Professor Benedikt M Kessler||Target Discovery Institute||Oxford University, NDM Research Building||GBRfirstname.lastname@example.org|
The linear ubiquitin chain assembly complex, LUBAC, is the only known mammalian ubiquitin ligase that makes methionine 1 (Met1)-linked polyubiquitin (also referred to as linear ubiquitin). A decade after LUBAC was discovered as a cellular activity of unknown function, there are now many lines of evidence connecting Met1-linked polyubiquitin to NF-κB signaling, cell death, inflammation, immunity, and cancer. We now know that Met1-linked polyubiquitin has potent signaling functions and that its deregulation is connected to disease. Indeed, mutations and deficiencies in several factors involved in conjugation and deconjugation of Met1-linked polyubiquitin have been implicated in immune-related disorders. Here, we discuss current knowledge and recent insights into the role and regulation of Met1-linked polyubiquitin, with an emphasis on the mechanisms controlling the function of LUBAC. Hide abstract
ISG15 is an interferon-induced and antiviral ubiquitin-like protein (Ubl). Herc5, the major E3 enzyme for ISG15, mediates the ISGylation of more than 300 proteins in interferon-stimulated cells. In addressing this broad substrate selectivity of Herc5, we found that: (1) the range of substrates extends even further and includes many exogenously expressed foreign proteins, (2) ISG15 conjugation is restricted to newly synthesized pools of proteins, and (3) Herc5 is physically associated with polyribosomes. These results lead to a model for ISGylation in which Herc5 broadly modifies newly synthesized proteins in a cotranslational manner. This further suggests that, in the context of an interferon-stimulated cell, newly translated viral proteins may be primary targets of ISG15. Consistent with this, we demonstrate that ISGylation of human papillomavirus (HPV) L1 capsid protein has a dominant-inhibitory effect on the infectivity of HPV16 pseudoviruses. Hide abstract
Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice. Hide abstract
Conjugation of Met1-linked polyubiquitin (Met1-Ub) by the linear ubiquitin chain assembly complex (LUBAC) is an important regulatory modification in innate immune signaling. So far, only few Met1-Ub substrates have been described, and the regulatory mechanisms have remained elusive. We recently identified that the ovarian tumor (OTU) family deubiquitinase OTULIN specifically disassembles Met1-Ub. Here, we report that OTULIN is critical for limiting Met1-Ub accumulation after nucleotide-oligomerization domain-containing protein 2 (NOD2) stimulation, and that OTULIN depletion augments signaling downstream of NOD2. Affinity purification of Met1-Ub followed by quantitative proteomics uncovered RIPK2 as the predominant NOD2-regulated substrate. Accordingly, Met1-Ub on RIPK2 was largely inhibited by overexpressing OTULIN and was increased by OTULIN depletion. Intriguingly, OTULIN-depleted cells spontaneously accumulated Met1-Ub on LUBAC components, and NOD2 or TNFR1 stimulation led to extensive Met1-Ub accumulation on receptor complex components. We propose that OTULIN restricts Met1-Ub formation after immune receptor stimulation to prevent unwarranted proinflammatory signaling. Hide abstract