An integrated study of the evolution, molecular function, expression and ubiquitome of the ligand of NUMB Protein X family

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dc.contributor.advisorYoung, Paul
dc.contributor.authorErwin, Leah G.en
dc.contributor.funderEli Lilly and Company
dc.contributor.funderUniversity College Cork
dc.date.accessioned2026-09-22T14:07:11Z
dc.date.available2026-09-22T14:07:11Z
dc.date.issued2026-03-31en
dc.date.submitted2026-03-31
dc.description.abstractThe LNX (Ligand of Numb Protein-X) / PDZRN (PDZ and RING) family are evolutionarily conserved E3 ubiquitin ligases characterised by a catalytic RING domain and multiple PDZ (protein binding) domains, originally identified through their interaction with the cell fate determinant NUMB. While there are several studies describing LNX proteins and their roles in vertebrate development, especially neuronal development in mice and dorso-ventral patterning in zebrafish, their broader physiological functions remain poorly understood. This is largely due to their extremely low endogenous expression levels and the complexity of their interaction networks. This thesis aims to provide a comprehensive depiction of LNX proteins, integrating bioinformatic, biochemical and experimental approaches. Evolutionary analyses in Chapter 5, demonstrate that LNX proteins maintain a highly conserved domain architecture across metazoan lineages, suggesting strong functional conservation. Structural modelling reveals that the second PDZ domain adopts an exposed conformation, explaining its promiscuity in mediating protein interactions, as confirmed in Chapter 3. However, specific interactions, such as the LNX-NUMB association mapped to the NPAY/F motif, appear to be vertebrate specific, indicating evolutionary neofunctionalisation. To further characterise the LNX protein interactome, a combination of yeast-two hybrid assays and affinity-purification based approaches were employed. While high-throughput datasets like ProfAff predict hundreds of potential interactors, experimental validation is necessary to reveal a more restricted set of physiologically relevant binding partners (Chapter 3 and Chapter 4). As an outcome of this approach, several novel interactions were identified, including neuronal associated proteins SDK2 and NRXN1β along with the transcriptional regulator ZNF24. To address challenges associated with low-abundance E3 ligases, new methodologies such as BioE3/Ub-POD were modified to identify LNX2 substrates. While proteomic analyses revealed diverse ubiquitination targets, interpretation of this data is complicated by apparent residual catalytic activity in the mutant LNX2 protein used as a control. Nevertheless, a list of putative novel LNX2 substrates was identified in Chapter 4. Several of these candidates were confirmed as binders of LNX PDZ domains, though further rigorous validation of their status as direct substrates of LNX2 ubiquitination is required. Lastly in Chapter 6, investigation of LNX1 and LNX2 expression showed that genomic amplification of LNX loci in colorectal and glial malignancies, does not appear to result in detectable protein levels in relevant cancer cell lines. LNX1 appears to act as a passenger gene in gliomas, whereas LNX2 displays expression patterns consistent with oncogenic drivers in colorectal cancer. Despite detectable mRNA and cell line transcriptomic data (Cancer Cell Line Encyclopaedia) suggesting RNA expression, endogenous protein levels remain low, suggesting tight post-transcriptional regulation. Promoter activity is generally weak but can be induced modestly under specific conditions like nutrient starvation, protein kinase C activation and drug treatments, indicating a role for LNX proteins in cellular stress and signalling adaptation. Overall, the data presented in this thesis provide novel functional and evolutionary insights regarding the enigmatic LNX1 and LNX2 ubiquitin ligases.en
dc.description.statusNot peer revieweden
dc.description.versionAccepted Versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.citationErwin, L. G. 2026. An integrated study of the evolution, molecular function, expression and ubiquitome of the ligand of NUMB Protein X family. PhD Thesis, University College Cork.
dc.identifier.endpage251
dc.identifier.urihttps://hdl.handle.net/10468/19309
dc.language.isoenen
dc.publisherUniversity College Corken
dc.relation.projectUniversity College Cork (School of Food Science and Engineering)
dc.rights© 2026, Leah Gabrielle Erwin.
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectLiganden
dc.subjectLNXen
dc.subjectProteinen
dc.subjectInteractionen
dc.subjectUbiquitinationen
dc.subjectE3 Ligaseen
dc.subjectPDZ domainen
dc.subjectRING domainen
dc.titleAn integrated study of the evolution, molecular function, expression and ubiquitome of the ligand of NUMB Protein X family
dc.typeDoctoral thesisen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD - Doctor of Philosophyen
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