Molecular Mechanisms of the Ubiquitin Ligase HERC2 in Cell Signalling: Implications in Cellular Stress, Autophagy and Proteasome Assembly
[eng] Ubiquitylation is a posttranslational modification that consists in the attachment of ubiquitin to a substrate protein. During the process of ubiquitylation, the E3 ubiquitin ligases determine the specificity of the substrates, thus, they represent a crucial regulatory factor. Among them is th...
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| Format: | doctoral thesis |
| Status: | Published version |
| Publication Date: | 2023 |
| Country: | España |
| Institution: | Universidad de Barcelona |
| Repository: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/219674 |
| Online Access: | https://hdl.handle.net/2445/219674 http://hdl.handle.net/10803/693988 |
| Access Level: | Open access |
| Keyword: | Proteïnes Ubiqüitina Estrès oxidatiu Autofàgia Proteins Ubiquitin Oxidative stress Autophagy |
| Summary: | [eng] Ubiquitylation is a posttranslational modification that consists in the attachment of ubiquitin to a substrate protein. During the process of ubiquitylation, the E3 ubiquitin ligases determine the specificity of the substrates, thus, they represent a crucial regulatory factor. Among them is the ubiquitin ligase HERC2, which belongs to the Large HERC protein family. HERC2 has been extensively studied for its implication in the regulation of genomic stability and p53 transcriptional activity. However, its involvement in other signalling pathways in which ubiquitylation also plays an important role remains fairly unexplored. Regarding the clinical relevance of HERC2, mutations in HERC2 gene in humans have been related to different pathologies such as a neurodevelopmental disorder known as HERC2 Angelman-like syndrome and several types of cancers. A better understanding of how pathologic HERC2 mutant variants affect intracellular signalling may aid definition of potential new therapies for these disorders. In view of all this, the main objective of this thesis was to study the signalling pathways regulated by HERC2 and to characterize the molecular mechanisms behind. We focused on the MAPK and autophagy pathways, and analysed how these are deregulated in pathological contexts of HERC2 deficiency. For that, we studied patient-derived fibroblasts of individuals with the HERC2 Angelman-like syndrome, which present a marked reduction in HERC2 protein levels. We complemented the investigations using other human and mouse cellular models by knocking down HERC2 protein expression. On the other hand, we applied a proteomic approach to identify ubiquitylated proteins regulated by HERC2, which can help to unveil novel physiological functions of this ubiquitin ligase. First, we studied MAPK signalling pathways in the patient-derived fibroblasts and observed increased levels of C-RAF protein and p38 phosphorylation. We showed that this occurs due to the fact that HERC2 regulates ubiquitin-mediated proteasomal degradation of C-RAF. Thus, in a context of HERC2 deficiency, C-RAF protein levels are upregulated. We found out that this upregulation triggers overactivation of a crosstalk between C-RAF and MKK3/p38 signalling pathways, which boosts the cellular response to oxidative stress through NRF2 and the expression of antioxidant genes. Secondly, we focused on autophagy and observed that it was increased in HERC2 deficient cells. Moreover, we demonstrated that HERC2 regulates protein levels of the autophagy initiating kinase ULK1 and the deubiquitinating enzyme USP20, which stabilizes ULK1. Additionally, we showed that HERC2 interacts with USP20, and observed that this interaction is disrupted upon p38 phosphorylation. Taken together these results suggested that HERC2 regulates autophagy through modulating USP20/ULK1 axis and that this process might be fine-tuned by p38 activity. In third place, we applied a proteomic approach to identify potential HERC2 ubiquitylation substrates. Results showed a functional enrichment of proteins related with the proteasome assembly pathway such as PSMC5 and PAAF1. We further confirmed that HERC2 interacts with these proteins in pull-down experiments. Finally, we discussed the molecular mechanisms of HERC2 in the regulation of MAPK signalling, autophagy and the proteasome assembly pathway, trying to postulate a working model that adds the results obtained to the knowledge stablished so far in the scientific literature. In addition, we inquired about how these mechanisms fit with the pathophysiology of the HERC2 Angelman-like syndrome and cancer. In conclusion, throughout this work we revealed previously unexplored functions of the ubiquitin ligase HERC2 in cell signalling. In particular, we described HERC2 as a regulator of: 1) the cellular response to oxidative stress through the C-RAF/MKK3/p38 pathway; 2) autophagy through the USP20/ULK1 axis; and 3) the proteasome assembly pathway through regulating ubiquitylation of proteins such as PSMC5 and PAAF1. |
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