Characterising Fibrillin Microfibril Structural Diversity and Photo-induced Damage

Student thesis: Phd

Abstract

Fibrillin microfibrils are supramolecular, extracellular matrix assemblies which play a number of important roles in different tissues. In eye, they exist as ciliary zonules which are inherent to the process of lens accommodation. By contrast, in skin dermis, they exist both as constituents of elastic fibres, endowing tissue with elasticity, and also as stand-alone oxytalan fibres, which extend through the papillary dermis. Although these assemblies play distinct key roles within these two tissues, it remains unknown whether their ultrastructure and molecular composition is tissue-dependent as a result. Fibrillin microfibrils have also been implicated in skin photoageing, which is characterised by the degeneration of its dermal architecture. Additionally, irradiation with physiological doses of ultraviolet (UV) radiation causes quantifiable changes to its beads-on-a-string ultrastructure, in vitro. As a result, the UV-susceptibility of this assembly has been implicated in the photoageing process. Although its ultrastructure is susceptible to UV, the molecular foci of this damage within the fibrillin-1 monomer remain undetermined. Also, in contrast to fibrillin, collagen VI microfibrils remain remarkably unaffected both by photoageing in skin and by UV irradiation. The molecular reasons behind this differential susceptibility also remain unknown. To address these gaps in knowledge, the first aim was to develop a novel mass spectrometry (MS)-based proteomic approach capable of characterising microfibril composition and monomeric structure of both fibrillin and collagen VI microfibril assemblies. Two digestion methods were successfully optimised: the elastase method led to a high fibrillin-1 primary coverage and revealed protein regions previously uncharacterised by proteomics. The SMART Digestion™ method also led to the identification of numerous co-purifying microfibril-associated proteins. Using this developed proteomic approach and atomic force microscopy (AFM), the next aim was to characterise tissue-dependent ultrastructural and compositional differences between skin-, eye- and human dermal fibroblast (HDF)-derived fibrillin microfibrils. This work revealed that, not only did the ultrastructure of eye fibrillin microfibrils differ from skin, but also tissue-specific regions of the fibrillin-1 structure were differentially susceptible to proteolysis. Furthermore, this work found that skin and HDF fibrillin microfibrils also differed in ultrastructure and fibrillin-1 proteolytic susceptibility. In contrast, collagen VI microfibrils from the same samples were invariant. Finally, it was observed that fibrillin microfibrils isolated from skin and eye exhibited unique protein compositions mirroring tissue function. This study showcased the diversity of this key assembly. Finally, this PhD aimed to discover which regions of the fibrillin-1 monomer were susceptible to UV and how this differs to the collagen VI alpha-3 (COL6A3) monomer. HDF fibrillin microfibril ultrastructure was significantly altered by both solar simulated radiation (SSR; 30 J/cm2) and UVB irradiation (100 mJ/cm2) whereas, comparatively, collagen VI microfibril ultrastructure was not. UV irradiation consistently enhanced the total yield of digested fibrillin-1 peptide, but not COL6A3. Mapping these peptides revealed that UV exposure increases regional susceptibility within the fibrillin-1 structure to proteolysis, allowing the identification of UV-susceptible foci on a molecular scale. Data-dependent quantification also revealed two fibrillin-1 peptides and ten COL6A3 peptides from skin isolations which matched UV-signatures also identified in HDF isolations indicating that this damage may also translate to microfibrils in native skin. This PhD successfully characterised fibrillin microfibril structural diversity and photo-induced molecular damage. In doing so, it has made a significant contribution to the field of ECM biology and photobiology, with the prospec
Date of Award1 Aug 2019
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorMichael Sherratt (Supervisor) & Rachel Watson (Supervisor)

Keywords

  • protein structure
  • ultraviolet
  • atomic force microscopy
  • ageing
  • photodamage
  • proteomics
  • mass spectrometry
  • eye
  • fibrillin
  • collagen VI
  • fibroblast
  • extracellular matrix
  • skin
  • microfibril

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