Many studies have demonstrated a connection between the fibrillin matrix and TGFβ signalling, but at present the mechanistic basis for this link is unclear. An interaction between the C-terminus of Latent TGFβ Binding Protein 1 (LTBP1) and the N-terminus of fibrillin1 has previously been identified, and may have the potential to directly link the fibrillin matrix to TGFβ signalling. To investigate the structural basis for this interaction, several multi-domain fragments of fibrillin1 and LTBP1 were expressed prokaryotically and refolded in vitro. After initial characterisation to confirm folding, the structure, dynamics, and interdomain interactions of these fragments were investigated in more detail using NMR techniques. Domains in both LTBP1 and fibrillin1 appear to demonstrate folds consistent with homologous structures, and while the LTBP1 C-terminal cbEGF14-TB3-EGF3-cbEGF15 region contains many flexible linkers and few interdomain interactions, the fibrillin1 EGF2-EGF3-hyb1-cbEGF1 region appears rigid, with interfaces forming between all domains present. SPR studies were used to demonstrate binding between distinct LTBP1 and fibrillin fragments, suggesting interactions between multiple domains are involved in the LTBP1-fibrillin1 interaction. The binding sites involved were then mapped to specific residues using HSQC titration studies, and structural models for the LTBP1-fibrillin1 interaction were generated based on these data. Predictions from these models were used to target residues for site-directed mutagenesis, based on their potential involvement in salt bridges, and when certain residues were replaced with those of opposite charge, reductions in binding could be seen in the SPR assay. These key residues were consistent with a particular model of the LTBP1-fibrillin1 interaction, as derived from the HSQC titration data. The conservation of potential binding site residues through deuterostome evolution also supports an important biological role for the LTBP-fibrillin interaction.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22134号 / 生博第421号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 上村 匡, 教授 荒木 崇, 教授 井垣 達吏 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Disruptions in cardiac development cause congenital heart disease, the most prevalent and deadly congenital malformation. Genetic and environmental factors are thought to contribute to these defects, however molecular mechanisms remain largely undefined. Recent work highlighted potential roles of chromatin- modifying enzymes in congenital heart disease pathogenesis. Histone deacetylases, a class of chromatin-modifying enzymes, have developmental importance and recognized roles in the mature heart. This thesis aimed to characterize functions of Hdac3 in cardiac development. We found loss of Hdac3 in the primary heart field causes precocious progenitor cell differentiation, resulting in hypoplastic ventricular walls, ventricular septal defect, and mid- gestational lethality. In primary heart field progenitors, Hdac3 interacts with, deacetylates, and functionally suppresses transcription factor Tbx5. Furthermore, a disease-associated Tbx5 mutation disrupts this interaction, rendering Tbx5 hyperacetylated and hyperactive. By contrast, deletion of Hdac3 in second heart field progenitors bypasses these defects, instead causing malformations in the outflow tract and semilunar valves, with lethality prior to birth. Affected semilunar valves and outflow tract vessels exhibit extracellular matrix and EndMT defects and activation of the Tgfβ1 signaling pathway. In normal second heart field development, Hdac3 represses Tgfβ1 transcription, independent of its deacetylase activity, by recruiting the PRC2 methyltransferase complex to methylate the Tgfβ1 promoter. Importantly, knockouts of Hdac3 in differentiated cardiac cells do not fully recapitulate the progenitor-specific knockout phenotypes. These results illustrate spatiotemporal roles of Hdac3, both deacetylase-dependent and deacetylase-independent, in cardiac development, suggesting that dysregulation of Hdac3 in cardiac progenitor cells could be a contributing factor in congenital heart disease pathogenesis.

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Vascular remodelling due to excessive proliferation and apoptosis resistance of pulmonary arterial smooth muscle (PASMCs) and endothelial cells (ECs) has been attributed to the pathogenesis of pulmonary arterial hypertension (PAH). It is an incurable cardiovascular disorder, which leads to right heart failure and death, if left untreated. Heterozygous germline mutations in the bone morphogenetic protein receptor type II (BMPR2) have been linked with the majority (~75%) of the familial form of the disease (HPAH). Mutations in the BMPR2 gene impinge upon the BMP signalling which perturbs the balance between BMP and TGF-β pathways leading to the clinical course of the disease. Current therapies were discovered prior to the knowledge that PAH has substantial genetic components. Hence, this study aims to identify novel therapeutic intervention and provide novel insights into how the dysfunctional BMPRII signalling contributes to the pathogenesis of PAH. This work demonstrates that cryptolepines and FDA approved drugs (doxorubicin, taxol, digitoxin and podophyllotoxin) inhibit the excessive proliferation and induce apoptosis in BMPR2 mutant PASMCs by modulating the BMP and TGF-β pathways. Moreover, established drug PTC124 has also been tested but has failed to promote translational readthrough. I have also shown that dysregulated apoptosis of PASMCs and HPAECs is mediated through the BMPRII-ALK1-BclxL axis. Finally, the siRNA screen targeting approximately 1000 genes has identified novel proteins including PPP1CA, IGF-1R, MPP1, MCM5 and SRC each capable of modulating the BMPRII signalling. Taken together, this study for the very first time has identified novel compounds with pro-BMP and anti-TGFβ activities which may provide therapeutic intervention prior to or after the onset of PAH. / Commonwealth Scholarship Commission in the UK / The full text will be available at the end of the embargo period, 31st July 2024.

Idiopathic Pulmonary Fibrosis (IPF) is a debilitating respiratory disorder that is characterized by a progressive decline in lung function. Originating through unknown etiology, it is essentially an unchecked wound healing response that causes the build-up of excessive scar tissue in the lung interstitial tissue with a heavy toll on the patient’s respiratory capacity. Pro-fibrotic alternatively activated macrophages (M2) have been linked as an important contributor to the fibrotic remodeling of the lung. Previous Ask research indicates that targeting M2 macrophages is possible through the use of the Dectin-1 receptor, a transmembrane cell surface receptor found in high abundance on M2 macrophages. Activating the Dectin-1 receptor through the use of beta-glucan, a ligand the receptor has a high affinity for, initiates a pro-inflammatory response within the naturally immunosuppressive macrophage and can alter its activity to be less fibrogenic. Our data suggest that M2 polarization of naïve macrophages can be inhibited in vitro by beta-glucan microparticles. Additionally, we have found that polarized M2 macrophages adopt M1-like characteristics when treated with beta-glucan microparticles, in a process that is largely Dectin-1 dependent. M2 cell surface marker CD206, increased levels of which are associated with rapidly progressing IPF, shows significantly decreased frequency of expression in M2 macrophages treated with beta-glucan microparticles. Our assessment for cell-specific uptake of beta-glucan microparticles suggests an important role of the Dectin-1 receptor for significantly increased uptake in murine wild-type M2 macrophages relative to their Dectin-1 knockout counterpart. The use of beta-glucan microparticles as a potential anti-fibrotic therapeutic was assessed in the bleomycin model of fibrotic lung disease. Mice given bleomycin and treated with beta-glucan displayed decreased soluble collagen content and TGFB expression within lung homogenate relative to fibrotic bleomycin control mice. Overall, these results provide insight into the use of beta-glucan as a potential activity modulator of macrophage function in IPF and the possibility of its use as a therapeutic. / Thesis / Master of Science (MSc)

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