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Browsing by study line "Biochemistry and Structural Biology"

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  • Mattila, Saku (2023)
    Plants are vital to all terrestrial ecosystems by providing ecosystem services through photosynthesis- derived compounds. Throughout the millennia, plant metabolism has diversified in the form of all plant secondary metabolites, ranging from metabolite groups such as terpenes to alkaloids to flavonoids. Many of these secondary metabolites are economically valued for their chemical, pharmaceutical and physical properties. The flavonoids are one of the largest groups and are known to provide competitional advantages and increase of survival of many plant species in extreme environments. One of the critical enzymes in the whole biosynthesis pathway of flavonoids is the dihydroflavonol 4- reductase (DFR). DFR regulates the formation of leucoanthocyanidins, predecessors of colourful anthocyanins. Anthocyanins are an economically significant group of molecules, especially for horticulturists and plant breeders, but also for nutritional and health scientists due to their potential health benefits. Dihydroflavonol 4-reductase is a much-studied enzyme due to its significant role in flavonoid biosynthesis and the economic interests of plant breeders and alike. Previous studies have expanded the knowledge of flavonoid biosynthesis and have identified several amino acid residues in the DFR structure affecting the substrate specificity of the enzyme and, consequently, the flower colours. However, only a single crystal structure model of the dihydroflavonol 4-reductase has been solved so far, originating from the grapevine Vitis vinifera. Although a single crystal structure can facilitate further structure-to-function studies associated with dihydroflavonol 4-reductase, further studies need to be carried out to shine a light on the functional basis of the enzyme. Therefore, this study aims to resolve petunia and gerbera dihydroflavonol 4-reductase crystal structures, expanding the knowledge of structural variations within the uncharted families of angiosperms, Solanaceae and Asteraceae. Several recombinant protein expression systems were utilised in my attempts to solve the crystal structure of the DFRs. These systems ranged from the bacterium Escherichia coli to yeast species such as Saccharomyces cerevisiae and Pichia pastoris, as well as the tobacco plant Nicotiana benthamiana. The genes encoding for Petunia wildtype DFRA, three mutants, and three Gerbera DFR variants were cloned to several expression vectors. Their presence and expression were identified using various genetic methodologies and enzymological assays. The expression of DFRs using an E. coli-based expression system was verified. However, the trials with E. coli were deemed unsuccessful due to the majority of the protein ending in inclusion bodies with no detectable activity. An alternative system using agroinfiltration of N. benthamiana was later utilised, as significant amounts were detected in the plant tissue extracts following the agrobacterial infiltration. Although the proteins were expressed in high quantities, no purification procedures have been established to provide plant tissue-extracted protein in crystallography-grade purity. With the protein supplied by a plant-based system and several small- scale purification steps, purified DFR enzymes could be utilised in crystallisation studies. Due to significant contamination by RuBisCO in the protein samples, alternative systems based on S. cerevisiae and Pichia pastoris were investigated, and a successful Pichia-based expression was established. Several sets of plasmids with variable expression systems were constructed in this study, facilitating future experiments into the dynamics and structure of dihydroflavonol 4-reductases. Ground-breaking techniques based on computational modelling were utilised to hypothesise the role of prior determined amino acid residues in enzyme catalysis and substrate recognition. Possible crystallisation-related issues originating from protein structure were approached using the same techniques, opening new windows and possibilities into determining the structure of Petunia hybrida and Gerbera hybrida dihydroflavonol 4-reductase structures using tools of protein engineering.
  • Tiilikainen, Emmi (2023)
    Lymphatic vascular system consists of lymphatic capillaries and collectors existing alongside a circulatory system of blood vessels. The lymphatic system is responsible of draining tissue fluids, trafficking of immune cells and intestinal absorption of dietary lipids. Most of the lymphatic networks develop during embryogenesis, but lymphangiogenesis (the growth of new lymphatic vessel, LV) occurs also in adult tissues, for example, during inflammation. Exposure to vascular endothelial growth factor C (VEGF-C) initiates lymphatic endothelial cell (LEC) proliferation and sprouting of LVs. In lymphangiogenesis, leading tip cell migrates and samples the surrounding environment while stalk cells proliferate and are responsible of LV elongation and extension. Since polarity of dividing cells and subsequent daughter cell positioning possess a key role in morphogenesis of tubular organs, such as lungs, kidney or blood vessels, a regulation of daughter LEC positioning after cell division might determine how LVs elongate and widen. The aim of this study was to investigate the LV network enlargement and daughter LEC positioning during growth of LVs and to reveal potential contributing factors guiding the cell positioning (such as cell polarity). In this study, the LV network of mouse ear pinna was used as a model tissue to investigate LV network enlargement, daughter LEC positioning and LEC polarity in growing LVs. Characterization of mitotic cells in developing LV network revealed that LEC proliferation occurs throughout the entire length of LVs in the network. To investigate LEC polarity in developing and mature LVs, I analysed Golgi and nuclear polarity of tip and stalk LECs. I found that whereas LECs during development are polarized and oriented along the long axis of LV, there is more variation in the direction of LEC polarity in relation to LV long axis in mature LV. This observation raised a question whether changes in the cell polarity were reflected to cell positioning, hence I analysed the positioning of daughter LECs by forcing LECs to the cell cycle with VEGF-C. These results indicated cell-level mechanisms that may contribute to LEC positioning in lymphangiogenesis. My finding provides an efficient tool for further research due to its suitability for monitoring proliferating LECs and studying causative factors affecting LEC proliferation and positioning. Future experiments with real-time imaging will reveal more about lymphangiogenesis process and provide insights into the role of lymphatic vasculature in conditions such as inflammation-related lymphedema or anti-tumor immunity in cancer.
  • Vainio, Jere (2022)
    Anthocyanins are pigment molecules responsible for the majority of flower colors existing in nature. Emerging from the flavonoid biosynthetic pathway, anthocyanin biosynthetic pathway branches into orange pelargonidin derivates, red cyanidin derivates and blue delphinidin derivates. Dihydroflavonol 4-reductase (DFR), a NADPH-dependent oxidoreductase, catalyzes the first anthocyanin specific step after the branching point for all three branches. In some cases, DFR exhibits substrate specificity leading to some flowering plant species’ inability to produce certain colors; like petunias lacking orange colors. Ornamental plant industry thrives on breeding of novel colors and color patterns, and thus understanding of the capabilities of anthocyanin biosynthesis is of key importance. The aim of this study is to gain insight into the amino acid residues causing substrate specificities in Petunia hybrida. The study focused on an amino acid region that has been previously identified as affecting substrate specificities in Gerbera hybrida. To examine the effects of three different mutations in this region, enzyme activity was examined both in vitro and in vivo. Experiments consisted of kinetic assays with protein extracts from infiltrated Nicotiana benthamiana and determination of anthocyanin content from stable transformations of Petunia hybrida. Anthocyanin content was determined from transformed petunia flowers with high performance liquid chromatography. Kinetic assays show distinct substrate specificity profiles for all three mutations, indicating a correlation between the studied residues and substrate specificity. The transformed petunias also exhibited altered anthocyanin content, with two of the three mutant transformants exhibiting increased pelargonidin production. The observed effects of these mutations support the previous results indicating that this region has a role in determining substrate specificities of DFR enzymes.
  • Nordlin, Patric (2023)
    The discovery and development of new antifungal drugs has lagged behind the clinical needs for effective treatments of fungal infections. Invasive fungal infections can be challenging to treat and can become life-threatening, particularly for immunosuppressed individuals. Despite the need for new and improved antifungal drugs, the pipeline for antifungal drug development has been relatively slow, with only a few new agents being approved in recent years. Many existing antifungal drugs have toxic side effects, limiting their use and highlighting the need for more targeted and effective therapeutics. The glycosylphosphatidylinositol biosynthesis pathway has been proposed as a potential new target for antifungal drugs. The glycosylphosphatidylinositol (GPI) anchor is a complex glycoconjugate that is attached to many proteins found on the surface of eukaryotic cells. GPI anchored proteins play important roles in various cellular processes, including signaling, cell adhesion, and cell recognition. The biosynthesis of GPI anchors involves multiple enzymatic steps, including the transfer of the GPI anchor to a target protein. Gpi3 is one of the key enzymes involved in the first step of GPI biosynthesis and is the catalytic subunit of the GPI GlcNAc-PI synthesis complex. The naturally occurring molecule Jawsamycin has been shown to selectively inhibit fungal Gpi3 while not interfering with its human ortholog. However, the development and research of Jawsamycin and other potential inhibitors of the GPI synthesis pathway are hampered by the lack of structural data on the proteins involved in the pathway. This thesis aimed to express and purify functionally active Gpi3 as a recombinant fusion protein using the SUMO tag expression system, with the end goal of utilizing the protein for structural studies through crystallography to better understand the function of Jawsamycin. In this thesis, Gpi3 was successfully expressed and purified as a fusion protein. However, enzymatic activity of Gpi3 was not observed, additionally, the purification and stability of the Gpi3 fusion proteins were shown to be problematic and no crystal structure of the protein of interest was acquired. These results indicate that a different approach is needed to gain structural insights into the function and interaction between Gpi3 and Jawsamycin. A likely path forward is the purification of the whole GPI GlcNAc synthesis complex which could give more insights into the organization and function of both Gpi3 and Jawsamycin.
  • Salmelin, Natasha Emmie Astrid (2023)
    Triple Negative Breast Cancer (TNBC) has the worst prognosis among all breast cancer subtypes. The lack of hormonal receptors and Her2 expression makes targeting with hormone-based treatments or anti-Her2 antibodies ineffective. Furthermore, TNBCs exhibit the highest expression of the oncogene MYC which negatively affects immune cell function. Natural Killer (NK) cells target transformed cells like cancer cells and have demonstrated promising clinical efficacy as treatments for hematological malignancies. However, NK cells have not yet been as successful in treating solid cancers, like breast cancer. The mechanism behind the lack of efficacy is not well understood, and therefore studies elucidating the mechanism are critical for improving the efficacy of NK cell therapies. In this study, we show that MYC-overexpression by itself does not affect the NK cell cytotoxicity of TNBC cell lines, however, if the NK cell response is initiated through antibody-dependent cellular cytotoxicity (ADCC) then MYC expression inhibits NK cell-mediated killing. Many TNBC cell lines are resistant to classical NK cell cytotoxicity, which we show can be overcome with ADCC-inducing antibodies. MYC overexpression has an inhibitory effect in two out of three NK cell donors, when overexpressed in the presence of ADCC-enabling antibodies. This indicates some degree of heterogeneity in MYC regulation of ADCC-dependent cytotoxicity. Our results also demonstrate that when MYC is overexpressed in TNBC cell lines, NK cell activating ligands are downregulated on the tumor cell surface, which could explain the MYC-mediated inhibition of NK cells. This is consistent with other studies where MYC overexpression downregulates NK cell activating ligands in cancer cell lines and inhibits NK cell killing. Altogether, we demonstrate a functional role of MYC in the inhibition of ADCC-dependent NK cell cytotoxicity in TNBC. These findings could explain the inhibitory function of tumor cells on NK cells and provide the rationale for exploring MYC-overexpression as a biomarker for predicting a response of breast cancer patients to NK cell-based immunotherapies.
  • Jayachandran, Rupesh Balaji (2022)
    COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has thus far claimed over six million lives. Vaccines against SARS-CoV-2 have successfully mitigated severe disease and eased the burden on healthcare systems. Neutralizing antibodies play crucial roles both in vaccine derived immunity, and as drugs widely utilized in monoclonal antibody therapy. Neutralizing antibodies primarily target the spike protein, where most of the neutralizing epitopes are located in the receptor binding domain (RBD). Elucidating the sites of vulnerability to neutralization on SARS-CoV-2 can facilitate the development of therapeutics. 7A12 is a newly-discovered IgG antigen-binding fragment (Fab) isolated from a COVID-19 patient in Finland that can neutralize SARS-CoV-2 by targeting the spike protein. In this thesis, a complex of the Fab 7A12 with SARS-CoV-2 spike ectodomain trimer was studied using cryogenic electron microscopy (cryo-EM) single-particle analysis to elucidate the epitope of 7A12 and to gain insight into the neutralization mechanism of 7A12. Cryo-EM data of the complex revealed that Fab 7A12 can bind to both “open” and “closed” conformations of RBD. Rigid-body fitting of the spike trimer and Fab 7A12 models into the cryo-EM density indicates that 7A12 recognizes an epitope in the RBD which is mainly located outside the ACE2 binding site. Together, these results suggest that the 7A12 epitope belongs to class III of SARS-CoV-2 neutralizing epitopes located in the RBD, indicating that 7A12 could neutralize by sterically hindering ACE2 and by preventing the adjacent RBD from changing to ”up” conformation. Furthermore, our results revealed an overlap of 7A12 epitope with the putative binding site of heparan sulfate, a host factor of SARS-CoV-2 infection, which might contribute to neutralization. 7A12-RBD interface mapping delineated the residues comprising the epitope, which do not include mutants found in Beta, Gamma, and Delta variants, while four mutants were found in Omicron within the epitope indicating that 7A12 is likely to neutralize Beta, Gamma, and Delta variants of concern.