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Functional and Localization Studies of Human Kynurenine 3-Monooxygenase

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posted on 13.07.2016, 15:17 by Aisha Mahmod O. Swaih
Kynurenine 3-monooxygensae (KMO) is an outer mitochondrial membrane protein which plays a critical regulatory role in the kynurenine pathway (KP), catalysing the production of 3-hydroxykynurenine (3-HK). Increased KMO activity likely contributes to the excitotoxicity seen in neurodegenerative disorders by elevating the levels of the neurotoxic KP metabolites 3-HK and quinolinic acid. Studies employing models of Huntington’s disease (HD) have shown that inhibition of KMO is neuroprotective, making KMO a potential therapeutic target for this disorder. This study interrogates the subcellular localisation of human KMO and dissects the interaction between KMO and the huntingtin (HTT) protein, mutations in which cause HD. Confocal microscopy based co-localisation studies of KMO demonstrated that full length KMO (flKMO) was exclusively localised to the mitochondria when expressed in HEK293T cells. Notably, deleting a C-terminal portion of flKMO which contains a putative transmembrane domain mis-localised the remaining protein (tKMO) to other cellular compartments. Localization of flKMO to the outer mitochondrial membrane was further confirmed via transmission electron microscopy. To study potential interactions between flKMO and HTT in living cells, bimolecular fluorescence complementation (BiFC) assay was utilised, which is based upon reconstitution of split fluorescence proteins. The BiFC approach allowed visualisation and quantification of flKMO interaction with both WT HTT and soluble mHTT fragments at the mitochondria. The strength of this interaction is inversely correlated to the length of the HTT polyglutamine expansion. Increased mitochondrial sub-cellular localisation of BiFC HTT constructs was confirmed via microscopy. tKMO however did not interact with HTT via the BiFC system, indicating that the C-terminal region of flKMO is important for both mitochondrial localisation and protein interaction. In total, these data suggest that flKMO-HTT interactions at the mitochondria may be biologically significant and could play a role in regulating KMO activity, and that in HD this regulatory process is impaired.

History

Supervisor(s)

Giorgini, Flaviano

Date of award

01/07/2016

Author affiliation

Department of Genetics

Awarding institution

University of Leicester

Qualification level

Doctoral

Qualification name

PhD

Language

en

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