Assessing the physiological relevance and molecular mechanisms of non-canonical ubiquitylation
Lead Research Organisation:
UNIVERSITY OF DUNDEE
Abstract
Ubiquitin is a small protein that acts like a cellular "tag" or "label." When conjugated to other proteins, it sends messages that tell the cell what to do with them. Most commonly, ubiquitin marks damaged or unneeded proteins for disposal - but it can also direct proteins to move to different places in the cell, change how they function, or help the cell respond to stress.
While scientists have studied this process, also known as “ubiquitylation” for many years, recent findings suggest that it can occur in unexpected ways, on different amino acids, not just on the commonly studied lysine residues, but also serine and threonine residues. This less-understood type of ubiquitylation, also known as non-canonical ubiquitylation, could play crucial roles in controlling cellular pathways, especially in relation to protein quality control in human health and disease.
Our research focuses on the UBE2Q family of ubiquitin-conjugating enzymes (E2s) -UBE2Q1, UBE2Q2, and UBE2QL1 - which catalyse the attachment of ubiquitin to serine and threonine residues.
During the initial Future Leaders Fellowship (FLF) period, the project delivered major foundational advances by identifying key and unique structural features that confer serine/threonine ubiquitylation capability to UBE2Qs.
A central question remains about the biological pathways regulated by the UBE2Q family. We initially focus on one member of the UBE2Qs family, UBE2Q1. By overlaying proteomic and RNA-seq datasets with Gene Ontology enrichment analysis we discovered that that UBE2Q1 significantly impacts the JAK–STAT signalling pathway in HeLa cell lines.
We next focused on the identification of the molecular machinery that allow UBE2Q1 to regulate the JAK-STAT pathway. Cullin-RING ligases (CRLs) - the largest E3 family - were identified in the proteomic dataset as potential UBE2Q1 partners. Specifically, ten CRLs substrate receptors, including FEM1B, ZER1, and KLHDC10, were significantly stabilised in UBE2Q1 knockout cells, suggesting dependence on UBE2Q1 for their turnover. Notably ZER1 was previously reported as a specific CUL2 substrate adaptor for proteins with N-terminal glycine, such as JAK2.
In vitro reconstitution experiments further demonstrated that UBE2Q1 cooperates robustly with the CUL2 complex to ubiquitylate JAK2-GFP pseudosubstrate, with activity enhanced by CUL2 neddylation. Structural modelling predicted distinct UBE2Q1–RBX1 interacting hotspots which were validated via targeted mutagenesis to confirm their functional relevance for CUL2-dependent ubiquitylation.
Together, these findings establish UBE2Q1 as a bona fide CRL-associated E2 enzyme that drives the ubiquitylation and degradation of N-glycine–degron substrates, including JAK2.
Building on these discoveries, the renewal proposal outlines three major aims. Aim 1 will define the endogenous UBE2Q1 ubiquitylation machinery using proximity-dependent biotinylation (mTurboID), cross-linking mass spectrometry, and complementary immunoprecipitation approaches. Aim 2 will map physiological UBE2Q1 substrates in vivo by enriching ubiquitylated proteins from WT and UBE2Q1 knockout cells using TUBEs, MultiDsk, and UbiSite, followed by proteomic identification of modified sites. Aim 3 will determine the structural basis of UBE2Q1-mediated serine/threonine ubiquitylation. A stable UBE2Q1–Ub conjugate will be generated via a C351K mutant to enable crystallisation and structural analysis with free threonine or threonine-containing peptides.
Together, this programme will define the molecular machinery, physiological roles, and structural principles of non-canonical ubiquitylation mediated by UBE2Q1.
While scientists have studied this process, also known as “ubiquitylation” for many years, recent findings suggest that it can occur in unexpected ways, on different amino acids, not just on the commonly studied lysine residues, but also serine and threonine residues. This less-understood type of ubiquitylation, also known as non-canonical ubiquitylation, could play crucial roles in controlling cellular pathways, especially in relation to protein quality control in human health and disease.
Our research focuses on the UBE2Q family of ubiquitin-conjugating enzymes (E2s) -UBE2Q1, UBE2Q2, and UBE2QL1 - which catalyse the attachment of ubiquitin to serine and threonine residues.
During the initial Future Leaders Fellowship (FLF) period, the project delivered major foundational advances by identifying key and unique structural features that confer serine/threonine ubiquitylation capability to UBE2Qs.
A central question remains about the biological pathways regulated by the UBE2Q family. We initially focus on one member of the UBE2Qs family, UBE2Q1. By overlaying proteomic and RNA-seq datasets with Gene Ontology enrichment analysis we discovered that that UBE2Q1 significantly impacts the JAK–STAT signalling pathway in HeLa cell lines.
We next focused on the identification of the molecular machinery that allow UBE2Q1 to regulate the JAK-STAT pathway. Cullin-RING ligases (CRLs) - the largest E3 family - were identified in the proteomic dataset as potential UBE2Q1 partners. Specifically, ten CRLs substrate receptors, including FEM1B, ZER1, and KLHDC10, were significantly stabilised in UBE2Q1 knockout cells, suggesting dependence on UBE2Q1 for their turnover. Notably ZER1 was previously reported as a specific CUL2 substrate adaptor for proteins with N-terminal glycine, such as JAK2.
In vitro reconstitution experiments further demonstrated that UBE2Q1 cooperates robustly with the CUL2 complex to ubiquitylate JAK2-GFP pseudosubstrate, with activity enhanced by CUL2 neddylation. Structural modelling predicted distinct UBE2Q1–RBX1 interacting hotspots which were validated via targeted mutagenesis to confirm their functional relevance for CUL2-dependent ubiquitylation.
Together, these findings establish UBE2Q1 as a bona fide CRL-associated E2 enzyme that drives the ubiquitylation and degradation of N-glycine–degron substrates, including JAK2.
Building on these discoveries, the renewal proposal outlines three major aims. Aim 1 will define the endogenous UBE2Q1 ubiquitylation machinery using proximity-dependent biotinylation (mTurboID), cross-linking mass spectrometry, and complementary immunoprecipitation approaches. Aim 2 will map physiological UBE2Q1 substrates in vivo by enriching ubiquitylated proteins from WT and UBE2Q1 knockout cells using TUBEs, MultiDsk, and UbiSite, followed by proteomic identification of modified sites. Aim 3 will determine the structural basis of UBE2Q1-mediated serine/threonine ubiquitylation. A stable UBE2Q1–Ub conjugate will be generated via a C351K mutant to enable crystallisation and structural analysis with free threonine or threonine-containing peptides.
Together, this programme will define the molecular machinery, physiological roles, and structural principles of non-canonical ubiquitylation mediated by UBE2Q1.
Organisations
People |
ORCID iD |
| Virginia De Cesare (Principal Investigator / Fellow) |