Characterisation of the role of the NEDD8 E3 ligase DCNL5 in the apoptosis response

Hsia, Oliver (2020) Characterisation of the role of the NEDD8 E3 ligase DCNL5 in the apoptosis response. PhD thesis, University of Glasgow.

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Abstract

Defective in cullin neddylation-like (DCNL) proteins are known to coordinate the addition of NEDD8 to the cullin subunit of the largest family of ubiquitin E3 ligases, the Cullin-RING ligases (CRLs), in a process known as neddylation. The human genome encodes five DCNL proteins which are thought to exhibit a large degree of overlap in function, with only a few neddylation processes having been definitively ascribed to a single DCNL homologue. It currently remains unclear whether these DCNL proteins have functions that extend beyond their roles in cullin neddylation. In the present study we now describe a novel role for one of the family members, DCNL5, in the programmed cell death response known as apoptosis. We have shown that cells lacking DCNL5 function fail to promote caspase 8 cleavage – an important early activation step - in response to various inducers of the extrinsic apoptosis pathway.

Caspase 8 cleavage and activation requires polyubiquitination which is known to be mediated by cullin 3 in coordination with the dual ubiquitin and NEDD8 ligase RBX1. This process is thought to occur in lipid rafts at the plasma membrane and in the cytosol. In the present work, we provide the first indication that DCNL5 is able to translocate out of the nucleus where it was previously thought to be exclusively located, and this occurs in response to TNFα-related apoptosis-inducing ligand (TRAIL) stimulation. In addition, we present evidence for the first known interaction between DCNL5 and cullin 3 in U2OS cells under endogenous conditions. The DCNL5 KO cells demonstrated a lack of a polyubiquitination event that occurs in WT cells; unmodified caspase 8 was shown to associate with a polyubiquitinated protein (the identity of which we were unable to determine) in response to TRAIL and this interaction was absent in KO cells, perhaps representing the key mechanism underlying DCNL5 involvement.

This emerging function for DCNL5 in promoting caspase 8 cleavage was confirmed in multiple cancer cell lines including U2OS, H460 and HeLa cells. Importantly, we demonstrated that siRNA-mediated silencing of DCNL5 prevented CASP8 cleavage. A lot of our work suggested that DCNL5’s role in CASP8 activation is mediated by the cullin CUL3. However, treatment with the neddylation inhibitor MLN4924 caused a reduction, but not a total loss of caspase 8 cleavage, suggesting that if cullin 3 is involved, it may be independent of its neddylation status. This hints at the surprising possibility that a CRL complex exists that that does not require neddylation for some of its function. Furthermore, while our data suggests that DCNL5 may regulate apoptosis via cullin 3, we were unable to exclude a cullin neddylation-independent role for DCNL5 in this process. Future work will need to answer this question by identifying and characterising the molecular target of DCNL5 in apoptosis signalling to ascertain the precise mechanism underlying DCNL5 regulation of this clinically important signalling event.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Supervisor's Name: Kurz, Dr. Thimo and Gould, Professor Gwyn
Date of Award: 2020
Depositing User: Mr Oliver Hsia
Unique ID: glathesis:2020-81680
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Sep 2020 13:47
Last Modified: 30 Sep 2020 13:47
URI: http://theses.gla.ac.uk/id/eprint/81680

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