Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.667958
Title: Characterisation of a novel interaction partner for Atg16L1 and its role in regulating autophagy
Author: Vaux, Laura C.
ISNI:       0000 0004 5364 2648
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2015
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Abstract:
Macroautophagy (hereafter referred to as autophagy) is a highly conserved eukaryotic cellular response to starvation, resulting in the formation of double-membrane vesicles known as autophagosomes. These sequester cytoplasmic contents to the lysosome, where they are degraded to generate amino acids which can be used for protein synthesis during periods of nutrient deprivation. Membrane targeting of LC3, the major structural protein of the autophagosome, is essential for autophagy. LC3 is processed from its cytosolic form (LC3-I) to its lipidated form (LC3-II) in a series of ubiquitin-like conjugation events. Of particular importance is Atg16-like-1 (Atg16L1), which through its interactions with Atg5-Atg12, specifies the site of LC3 incorporation into the expanding autophagosomal membrane. In this study tools have been generated for the study and characterisation of Atg16L1 domains in cell culture. Atg16L1 -/- MEFs have also been generated and used for reconstitution experiments, which confirm that the coiled-coil domain of Atg16L1 is sufficient to restore autophagy in response to starvation and to a surrogate pathogen. A novel interaction between Atg16L1 and the serine/threonine kinase MEKK4 has been characterised in this study. Existing research has established an interaction between the pathogen recognition receptors NOD1/2 and Atg16L1. The NOD receptors are activated by bacterial peptidoglycan (PG), which has been shown to drive autophagy. Interestingly, MEKK4 inhibits NOD2 signalling by binding the downstream adaptor protein RIP2, leading to the hypothesis that MEKK4 may play a role in autophagy. The interaction was verified by co-immunoprecipitation, with domain analysis indicating that MEKK4 binds to the linker region of Atg16L1. The kinase activity of MEKK4 was also required for the interaction. The complex between Atg16L1 and MEKK4 persisted during starvation induced autophagy. Lipidation of LC3 in response to starvation required expression of MEKK4, with a redistribution of LC3 to large perinuclear aggregrates that co-localised with p62 and ubiquitin in siRNA silenced cells and CRISPR/Cas MEKK4 knockout cells. The aggregates were also able to recruit LC3 I, indicating a role of aggresome-like induced structures (ALIS). This thesis documents an interaction between Atg16L1 and MEKK4 that is vital for LC3 processing. The potential role for MEKK4 in regulating autophagy through Atg16L1 is discussed and experiments proposed to further explore this interaction in the context of autophagy.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.667958  DOI: Not available
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