Browsing by study line "Biochemistry and Structural Biology"

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.

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.

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.