A microfibril assembly assay identifies different mechanisms of dominance underlying Marfan syndrome, stiff skin syndrome and acromelic dysplasias

Fibrillin-1 is the major component of the 10–12 nm diameter extracellular matrix microfibrils. The majority of mutations affecting the human fibrillin-1 gene, FBN1, result in Marfan syndrome (MFS), a common connective tissue disorder characterised by tall stature, ocular and cardiovascular defects. Recently, stiff skin syndrome (SSS) and a group of syndromes known collectively as the acromelic dysplasias, which typically result in short stature, skin thickening and joint stiffness, have been linked to FBN1 mutations that affect specific domains of the fibrillin-1 protein. Despite their apparent phenotypic differences, dysregulation of transforming growth factor β (TGFβ) is a common factor in all of these disorders. Using a newly developed assay to track the secretion and incorporation of full-length, GFP-tagged fibrillin-1 into the extracellular matrix, we investigated whether or not there were differences in the secretion and microfibril assembly profiles of fibrillin-1 variants containing substitutions associated with MFS, SSS or the acromelic dysplasias. We show that substitutions in fibrillin-1 domains TB4 and TB5 that cause SSS and the acromelic dysplasias do not prevent fibrillin-1 from being secreted or assembled into microfibrils, whereas MFS-associated substitutions in these domains result in a loss of recombinant protein in the culture medium and no association with microfibrils. These results suggest fundamental differences in the dominant pathogenic mechanisms underlying MFS, SSS and the acromelic dysplasias, which give rise to TGFβ dysregulation associated with these diseases

A possible role of Werner helicase interacting protein 1 (WRNIP1) in the Fanconi anemia DNA repair pathway

The Fanconi anemia (FA) pathway plays a crucial role in resolving DNA interstrand crosslinks (ICLs), which can form as a result of DNA damage by genotoxic agents (Lopez-Martinez et al., 2016). The exact mechanism of FA pathway action is unknown, although 21 proteins have been identified as its components, which, in turn, form at least three known groups, i.e. core, FANCD2/FANCI and downstream effector proteins (Mamrak et al., 2016; Ceccaldi et al., 2016). The FANCD2/FANCI complex is multi-phosphorylated and monoubiquitinated on both FANCD2 and FANCI after ICL-inducing damage. However, how FANCD2/FANCI complex is recruited to damaged chromatin remains elusive (Cohn and DâAndrea, 2008). This project investigates whether WRNIP1 can function as a protein operating in the Fanconi anemia pathway and examines its interaction with FANCD2 and FANCI. It was demonstrated here that WRNIP1 behaved similarly to the Fanconi anemia proteins because its depletion resulted in higher sensitivity of cells to the ICL-inducing agents, i.e. mitomycin C (MMC), TMP combined with UVA irradiation as well as cisplatin. Moreover, it was demonstrated that WRNIP1 partially rescued the sensitivity of WRNIP1-depleted cells after their complementation. It has been demonstrated that following treatment with genotoxic agents of cells and their subsequent fractionation, WRNIP1 did not show any significant increase in its nuclear fraction levels. Furthermore, WRNIP1-deficient cells treated with ICL-inducing agents and subsequently fractionated did not affect the ubiquitination patterns of FANCD2 nor did WRNIP1 depletion affect the formation of FANCD2 repair foci. These data might indicate that WRNIP1 was already present in the nucleus at the levels necessary to repair DNA. However, the lack of alteration in the FANCD2 ubiquitination and absence of effect of FANCD2 foci formation might suggest that WRNIP1 acted downstream of FANCD2 in the Fanconi anemia pathway or that there was no interaction between these two proteins. Therefore, this part of the project did not establish a functional link between FANCD2 and WRNIP1. It was demonstrated for the first time by in vitro binding analysis that WRNIP1 could bind to the two established components of the Fanconi anemia pathway, i.e. FANCD2 and FANCI. This result was corroborated by a subsequent study which revealed that FANCD2, FANCI and WRNIP1 could also form a complex together. It was shown that WRNIP1 bound to FANCD2 independently of FANCI. The in vitro binding analysis also attempted to map a WRNIP1âs domain which would be responsible for interaction with FANCD2. However, the three investigated WRNIP1 deletion mutants were shown to bind FANCD2. This outcome indicated the possibility that either WRNIP1 amino acid sequences located in between the three previously-generated regions were responsible for FANCD2-WRNIP1 interaction or, alternatively, WRNIP1 might bind to FANCD2 via two or more domains. It was also shown here that WRNIP1 was purified in two forms, one of which was demonstrated to be ubiquitinated. However, the type of ubiquitination has not been determined yet. It was demonstrated that deletion of WRNIP1's UBZ domain abrogates its ubiquitination but the consequences of this phenomenon must still be established.</p

Fabrication of an aptamer-functionalised silica nanoparticle construct and its separation by magnetic capture-hybridisation

Nanoparticles produced with surfaces functionalised by highly specific molecular tags are able to target aberrant cells and detect or eliminate them without causing damage to surrounding healthy tissues. Single-stranded DNA (ssDNA) and RNA which fold to form secondary or tertiary structures, termed aptamers, represent a new class of such molecular tags. The nanoparticles, in turn, may carry therapeutic payload or luminescent entities which enable elimination or visualisation of targeted cells respectively.This project presents fabrication and isolation of a surface-functionalised nanoparticle construct, namely aptamer-tagged silica nanoparticles. DNA aptamers were chosen with the intention to make them useful for clinical or diagnostic applications of targeting neoplastic cells. Indeed, the ssDNA applied here is known to bind mucin-1 which in turn is a biomarker found on the surface of metastatic breast cancer cells. The separation of the construct was made possible by the inclusion of oligonucleotide-bound superparamagnetic particles in the construct; these enabled separation by magnetic capture.This project investigates two approaches to fabrication of the construct. In the first approach, aptamers, oligonucleotides and magnetic particles are mixed in solution. In the second, silica nanoparticles are functionalised with aptamers, oligonucleotides are bound to magnetic particles and the resulting two parts are hybridised together. The first approach gives higher yields. This may suggest that binding of silica nanoparticles to aptamers may hinder aptamer hybridisation to oligonucleotide fragments, thus resulting in lower construct synthesis yields. However, it is not known yet how the yield changes upon addition of silica nanoparticles into the solution. Therefore, the second experimental approach provides a starting point for fabrication and purification of an anti-cancer drug targeting platform in a simple bench-top setting.In addition, this thesis discusses the fabrication of silica nanoparticles which were intended to constitute an element of the construct. The work on nanoparticle fabrication aimed to develop a quick and repeatable synthesis method which would result in monodisperse entities. Despite trying various experimental approaches, suitable particles could not be reproducibly obtained. Agglomeration was identified as a major obstacle in the silica nanoparticle production process.Finally, this project assesses whether the chosen aptamers bind to the metastatic breast cancer cells, which would be necessary if they were to be used for diagnosis or therapy. FACS analysis indeed indicate that ssDNA aptamers attach to the MCF7 cell line, but the optimum conditions for that attachment remain to be determined.</p

Fabrication of an aptamer-functionalised silica nanoparticle construct and its separation by magnetic capture-hybridisation

Nanoparticles produced with surfaces functionalised by highly specific molecular tags are able to target aberrant cells and detect or eliminate them without causing damage to surrounding healthy tissues. Single-stranded DNA (ssDNA) and RNA which fold to form secondary or tertiary structures, termed aptamers, represent a new class of such molecular tags. The nanoparticles, in turn, may carry therapeutic payload or luminescent entities which enable elimination or visualisation of targeted cells respectively.This project presents fabrication and isolation of a surface-functionalised nanoparticle construct, namely aptamer-tagged silica nanoparticles. DNA aptamers were chosen with the intention to make them useful for clinical or diagnostic applications of targeting neoplastic cells. Indeed, the ssDNA applied here is known to bind mucin-1 which in turn is a biomarker found on the surface of metastatic breast cancer cells. The separation of the construct was made possible by the inclusion of oligonucleotide-bound superparamagnetic particles in the construct; these enabled separation by magnetic capture.This project investigates two approaches to fabrication of the construct. In the first approach, aptamers, oligonucleotides and magnetic particles are mixed in solution. In the second, silica nanoparticles are functionalised with aptamers, oligonucleotides are bound to magnetic particles and the resulting two parts are hybridised together. The first approach gives higher yields. This may suggest that binding of silica nanoparticles to aptamers may hinder aptamer hybridisation to oligonucleotide fragments, thus resulting in lower construct synthesis yields. However, it is not known yet how the yield changes upon addition of silica nanoparticles into the solution. Therefore, the second experimental approach provides a starting point for fabrication and purification of an anti-cancer drug targeting platform in a simple bench-top setting.In addition, this thesis discusses the fabrication of silica nanoparticles which were intended to constitute an element of the construct. The work on nanoparticle fabrication aimed to develop a quick and repeatable synthesis method which would result in monodisperse entities. Despite trying various experimental approaches, suitable particles could not be reproducibly obtained. Agglomeration was identified as a major obstacle in the silica nanoparticle production process.Finally, this project assesses whether the chosen aptamers bind to the metastatic breast cancer cells, which would be necessary if they were to be used for diagnosis or therapy. FACS analysis indeed indicate that ssDNA aptamers attach to the MCF7 cell line, but the optimum conditions for that attachment remain to be determined.This thesis is not currently available via ORA

A microfibril assembly assay identifies different mechanisms of dominance underlying Marfan syndrome, stiff skin syndrome and acromelic dysplasias

Fibrillin-1 is the major component of the 10–12 nm diameter extracellular matrix microfibrils. The majority of mutations affecting the human fibrillin-1 gene, FBN1, result in Marfan syndrome (MFS), a common connective tissue disorder characterised by tall stature, ocular and cardiovascular defects. Recently, stiff skin syndrome (SSS) and a group of syndromes known collectively as the acromelic dysplasias, which typically result in short stature, skin thickening and joint stiffness, have been linked to FBN1 mutations that affect specific domains of the fibrillin-1 protein. Despite their apparent phenotypic differences, dysregulation of transforming growth factor β (TGFβ) is a common factor in all of these disorders. Using a newly developed assay to track the secretion and incorporation of full-length, GFP-tagged fibrillin-1 into the extracellular matrix, we investigated whether or not there were differences in the secretion and microfibril assembly profiles of fibrillin-1 variants containing substitutions associated with MFS, SSS or the acromelic dysplasias. We show that substitutions in fibrillin-1 domains TB4 and TB5 that cause SSS and the acromelic dysplasias do not prevent fibrillin-1 from being secreted or assembled into microfibrils, whereas MFS-associated substitutions in these domains result in a loss of recombinant protein in the culture medium and no association with microfibrils. These results suggest fundamental differences in the dominant pathogenic mechanisms underlying MFS, SSS and the acromelic dysplasias, which give rise to TGFβ dysregulation associated with these diseases