A structure-function analysis of Drosophila Tolloid

Members of the Tolloid (Tld) family of metalloproteinases play an essential role in Bone Morphogenetic Protein (BMP) gradient formation during embryonic dorsal-ventral axis patterning, by liberating BMPs from inhibitory complexes. Tolloid enzymes also play important roles during ECM processing. Howe...

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Main Author: Winstanley, Jennifer
Other Authors: Baldock, Clair ; Ashe, Hilary
Published: University of Manchester 2015
Online Access:https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.764370
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spelling ndltd-bl.uk-oai-ethos.bl.uk-7643702019-03-05T15:27:20ZA structure-function analysis of Drosophila TolloidWinstanley, JenniferBaldock, Clair ; Ashe, Hilary2015Members of the Tolloid (Tld) family of metalloproteinases play an essential role in Bone Morphogenetic Protein (BMP) gradient formation during embryonic dorsal-ventral axis patterning, by liberating BMPs from inhibitory complexes. Tolloid enzymes also play important roles during ECM processing. However, the mechanism and regulation of Tolloid is unclear. Using in vitro biophysical and biochemical approaches combined with in vivo analysis of Tolloid activity, this study aims to complete a structure-function analysis of Tolloid in relation to its activity in the Drosophila early embryo. The mechanism and regulation of Tolloid activity are dissected, with a specific focus on the roles of the non-catalytic CUB domains. In vitro activity analysis and binding assays identify a role for the N-terminal CUB domains of Tolloid in interaction with the extracellular matrix protein Collagen IV. This interaction enhances cleavage of the Tolloid substrate Sog. In contrast, the C-terminal CUB domains, CUB4 and CUB5, mediate interaction with substrate Sog. Electron Microscopy and Small Angle X-ray Scattering demonstrate that curvature of monomeric Drosophila Tolloid brings these Sog-binding CUB domains into close proximity to the N-terminal protease domain. Furthermore, the importance of these interactions is investigated in vivo using point mutation data from classical mutational analysis of Drosophila Tolloid. In addition, the BMP gradient is recapitulated in vivo in the absence of Tolloid CUB domains through artificially tethering the Tolloid protease domain to Sog. Extrapolation of the data presented here suggests that the dual use of CUB domains in substrate and ECM interactions may function to fine-tune Tolloid activity to a particular context.University of Manchesterhttps://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.764370https://www.research.manchester.ac.uk/portal/en/theses/a-structurefunction-analysis-of-drosophila-tolloid(9d089c82-1e26-4a32-a0ec-c3964df22c48).htmlElectronic Thesis or Dissertation
collection NDLTD
sources NDLTD
description Members of the Tolloid (Tld) family of metalloproteinases play an essential role in Bone Morphogenetic Protein (BMP) gradient formation during embryonic dorsal-ventral axis patterning, by liberating BMPs from inhibitory complexes. Tolloid enzymes also play important roles during ECM processing. However, the mechanism and regulation of Tolloid is unclear. Using in vitro biophysical and biochemical approaches combined with in vivo analysis of Tolloid activity, this study aims to complete a structure-function analysis of Tolloid in relation to its activity in the Drosophila early embryo. The mechanism and regulation of Tolloid activity are dissected, with a specific focus on the roles of the non-catalytic CUB domains. In vitro activity analysis and binding assays identify a role for the N-terminal CUB domains of Tolloid in interaction with the extracellular matrix protein Collagen IV. This interaction enhances cleavage of the Tolloid substrate Sog. In contrast, the C-terminal CUB domains, CUB4 and CUB5, mediate interaction with substrate Sog. Electron Microscopy and Small Angle X-ray Scattering demonstrate that curvature of monomeric Drosophila Tolloid brings these Sog-binding CUB domains into close proximity to the N-terminal protease domain. Furthermore, the importance of these interactions is investigated in vivo using point mutation data from classical mutational analysis of Drosophila Tolloid. In addition, the BMP gradient is recapitulated in vivo in the absence of Tolloid CUB domains through artificially tethering the Tolloid protease domain to Sog. Extrapolation of the data presented here suggests that the dual use of CUB domains in substrate and ECM interactions may function to fine-tune Tolloid activity to a particular context.
author2 Baldock, Clair ; Ashe, Hilary
author_facet Baldock, Clair ; Ashe, Hilary
Winstanley, Jennifer
author Winstanley, Jennifer
spellingShingle Winstanley, Jennifer
A structure-function analysis of Drosophila Tolloid
author_sort Winstanley, Jennifer
title A structure-function analysis of Drosophila Tolloid
title_short A structure-function analysis of Drosophila Tolloid
title_full A structure-function analysis of Drosophila Tolloid
title_fullStr A structure-function analysis of Drosophila Tolloid
title_full_unstemmed A structure-function analysis of Drosophila Tolloid
title_sort structure-function analysis of drosophila tolloid
publisher University of Manchester
publishDate 2015
url https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.764370
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