Browsing by study line "Biokemia ja rakennebiologia"

Plants have evolved mechanisms to cope with various environmental stresses, including abiotic factors like temperature extremes and biotic factors involving the interactions with pathogens and herbivores. Kale (Brassica oleracea var. acephala) is a superfood famous for containing many compounds that are beneficial in the human diet but are primarily produced as specialised metabolites to aid in plant defence. Amongst these are glucosinolates which are defence compounds characteristic of plants in the Brassicaceae family. The aim of this study was to investigate how the diverse metabolic profiles of kale cultivars contribute to postharvest resistance against herbivory and necrotrophy. To assess the resistance of each kale cultivar against herbivory, I used the larvae of the wood tiger moth Arctia plantaginis as a test subject. We used detached leaves from 30 kale cultivars in an overnight feeding experiment with the larvae. The same 30 cultivars were used in a postharvest infection experiment with a generalist necrotroph B. cinerea to investigate the resistance of each kale cultivar against necrotrophy. For a comparative experiment between necrotrophs, we selected 10 kale cultivars to assess the necrosis caused by B. cinerea and a specialist necrotroph A. brassicicola. The A. brassicicola-infected and mock-treated leaves were analysed for their metabolic profiles to observe how these were altered by the infection. The weight gain of the tiger moth larvae was not significantly affected by the kale cultivars or their sugar content. A correlation between sucrose and indole glucosinolates might have reduced the kales’ palatability and potentially deterred the herbivores. In the B. cinerea experiment, we observed a positive correlation between necrotic lesion area and protein, sucrose, and indole GSL contents in kale leaves, even though indole GSLs are generally considered defence molecules against necrotrophic pathogens. When comparing the necrotic damage caused by the two necrotrophs, the specialist A. brassicicola exhibited a statistically significantly more violent infection compared to B. cinerea. Chlorophyll became degraded in the infected leaves compared to the uninfected controls. Amino acid content was high in the aged control and infected leaves, indicating protein degradation either due to senescence or cell wall-degrading enzymes from the pathogen. There was a statistically significant positive correlation between necrotic damage and protein in the infected leaves potentially due to proteins being secreted by the pathogen during infection. Starch levels decreased in the infected leaves compared to the controls. The infected samples also showed decreased glucose amounts potentially being taken up by the necrotroph during infection. Altogether, the study showed that kale cultivars respond to biotic stress factors by triggering metabolic changes that can affect the disease resistance and postharvest quality of the leafy vegetables.

Intrinsically disordered proteins (IDPs) consist of charged, and polar amino acids, lacking bulky hydrophobic residues and they do not have a single well-defined 3D structure. They are found in all domains of life with higher abundance in eukaryotes, covering approximately 30% of the eukaryotic proteome. IDPs have key roles in many biological processes from cell signaling to phase-separation phenomena. Particularly, disordered protein regions serving as linkers, have been found in many multidomain proteins and they play a decisive role in the protein’s function. In the present thesis we aim to identify the correlation between sequence and rigidity disordered linkers, utilizing a synergistic method of Nuclear Magnetic Resonance (NMR) experiments and Molecular Dynamic (MD) simulations. For that purpose, glycine and proline rich disordered linkers which are widely utilized for constructing fusion proteins were used. Additionally, we aim to characterize the rigidity of the the disordered repetitive domain of the major ampullate type I dragline silk protein, using the same approach which served to connect the two terminal folded domains inside the protein. Dragline silk has been under thorough investigation due to its favorable mechanical properties and applications in material science. NMR spin relaxation times T1, T2 and hetNOE, are highly sensitive probes to motional timescales of IDPs, but they are difficult to interpret in terms of molecular dynamics. Here, we use the spin relaxation times to validate the MD simulations which in turn are set to interpret the linkers’ internal motions. Using the quality evaluation approach QEBSS, the best simulations were identified as the best description of the conformational ensemble, based on the comparison with the experimental spin relaxation times. Systematic differences in spin relaxation times correlate with systematic changes in the linkers rigidity, proving that spin relaxation times can be used to detect disordered linker rigidity. Prolines are shown to induce a comparatively expanded conformation ensemble with significantly slower dynamics whereas glycines offer flexibility. The ensemble of the repetitive domain of the silk protein showed conformations with intermediate rigidity. We also demonstrate that the synergy of NMR and MD simulations can be used for characterizing the rigidity-sequence interplay in short glycine and proline disordered linkers and silk protein systems. Being able to tune the properties of flexible and rigid linkers can be fundamental for understanding different biological systems and for protein engineering purposes. Bioengineering applications include designing and optimizing fusion protein linkers that in the long term be useful for drug design and developing protein-based biomaterials.

Parkinson’s disease (PD) is the second most common neurogenerative disease. There are no drugs available to halt the progression of PD. The glial cell line-derived neurotrophic factor (GDNF) has been identified as a potential drug candidate against PD because of its protective properties on dopaminergic neurons, which are an especially vulnerable cell population in PD. It has been recently shown that GDNF can also attenuate aggregation of phosphorylated α-synuclein in dopaminergic neurons, which is one of the most important pathologies of PD. Phosphorylated α-synuclein is a primary component of Lewy bodies, which in turn, are vastly studied intracellular inclusions with a high correlation towards neurodegenerative diseases. GDNF signals through its main receptor RET and activates downstream signalling cascades. RET is indispensable for the effect of GDNF against α-synuclein aggregation. Importance of the downstream molecules Src, AKT and PI3K have been also pharmacologically demonstrated. However, complete mechanism of GNDF’s action and individual importance of downstream signalling molecules has been yet to establish. CRISPR/Cas9 gene editing tool has revolutionized the gene manipulation in biological research. In this thesis work, CRISPR/Cas9 guides were designed to target and mutate the c-Src, Akt1 and NURR1, which are important proteins of the GDNF/RET pathway. As a delivery system for the Cas9 enzyme and individual guides, lentiviral vectors were produced according to the protocols previously established in our laboratory and proved to be high efficiency. Modelling of α-synuclein aggregation in neurons was performed with pre-formed fibrils of α-synuclein, which induce the formation of intracellular Lewy body-like inclusions with the phosphorylation of α-synuclein at serine 129. In this study, primary dopaminergic neuron cultures from E13.5 mouse embryos were cultured in 96-well plates. For each of the target genes, I designed two guide variants, cloned them in lentiviral transfer vectors and produced lentiviral particles for neuronal transduction. My data shows that targeting Akt1 and c-Src impaired the protective mechanism of GDNF against Lewy body-like inclusions. For the importance of NURR1 more studies are needed for coherent conclusions. I also showed that targeting of NURR1 impaired the GDNF/RET signalling at least in one guide construct. The 15-day long cultivation did not affect to the dopaminergic cell numbers in any of the groups. Still the confirmation of successful CRISPR-induced genetic mutations by sequencing as well as the detailed mechanism of how GDNF prevents the formation of Lewy body-like inclusions will be a subject of future studies. This thesis provides important information for the molecular mechanism of attenuation of α-synuclein aggregation by GDNF through its main receptor RET.

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.

Atherosclerosis is a cardiovascular disease characterized by the formation and growth of plaque within the arteries. Lipoproteins, especially LDL, initiate atherosclerosis by accumulating in the intima of arteries and becoming modified, e.g. oxidised. Oxidised LDL (OxLDL) is highly pro-atherogenic and promotes atherosclerosis in multiple ways. The role of platelets in the later stages of atherosclerosis is well-documented, but platelets may also be involved in earlier stages of atherosclerosis. Platelets release extracellular vesicles (PEVs) in the form of microvesicles (microparticles) and exosomes that participate in intercellular signalling and in similar pathophysiological processes as platelets. Lipoproteins are known to activate platelets but their effects on PEV formation have not yet been studied. The aim of this thesis was to investigate the effect of OxLDL on PEV formation and compare it to other potential agonists such as LDL, HDL, ATP, thrombin and collagen. Platelets were activated with these agonists separately or in combination with OxLDL. PEVs were studied from the platelet-depleted supernatant and the isolate, which was obtained by differential centrifugation. PEVs were quantified in terms of CD61+ PEVs and particle count by flow cytometry and nanoparticle tracking analysis, respectively. PEVs were characterized by the relative amounts of CD41 (platelet and PEV marker) and Hsp70 (general EV marker) detected by Western blotting. Lastly, the uptake of the differently induced PEVs by HepG2 hepatoma cells was compared by fluorescence microscopy as a characterization of the PEVs’ functionality. Among the lipoproteins, OxLDL was indicated to be a much more potent inducer of PEVs than LDL or HDL, as shown by flow cytometry of CD61+ PEVs, nanoparticle tracking analysis and CD41 and Hsp70 levels in the isolates. However, OxLDL was not as strong a PEV inducer as the co-stimulation with thrombin and collagen (T&C), which induced the highest PEV formation. Size distribution analysis showed that PEVs smaller than 100 nm in size comprised a larger proportion of the total PEVs in OxLDL-induced PEVs compared to LDL- and T&C-induced PEVs. OxLDL combined with weak PEV inducers such as HDL and ATP had an amplifying effect on the generation of CD61+ PEVs, while the highest PEV formation was observed when OxLDL was combined with thrombin and collagen. When OxLDL-induced PEV formation was tested against a range of HDL concentrations, the extent of PEV formation and relative Hsp70 levels both decreased in a HDL concentration-dependent manner up to 50 µg/mL HDL. Both LDL- and OxLDL-induced PEVs were taken up by HepG2 cells, but there was no statistically significant difference between the two. The results indicated the potency of OxLDL in inducing PEV formation, thereby suggesting a novel mechanism by which OxLDL could contribute to the progression of atherosclerosis. Further studies on OxLDL-induced PEVs are needed, but if significant lipoprotein-specific changes in PEV numbers and properties could be observed, PEVs could then be used as a biomarker to diagnose atherosclerosis already at the early stages.

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.

The infection mechanisms between cold-active bacteria and their respective bacteriophages are currently relatively unknown and undocumented. Shewanella sp. 4 is a cold-active bacterium that was recently isolated from Baltic Sea ice along with bacteriophage isolate 1/4. Little is known about this particular isolate, although many Shewanella species have important environmental roles incl. carbon cycling, and they have also been associated with the spoilage of fishery products and bioremediation. Previous studies have shown that an infection caused by bacteriophages may lead to significant changes in transfer RNA (tRNA) modifications in the host cell. Commonly, tRNA modification levels may be altered as a response to different stressors, to which viral infections belong as well. Bacteriophages may take advantage of tRNA modifications during the infection of their host, as changes in tRNA modifications lead to much faster response than affecting only the transcription and translation machineries. Here, the infection cycle and changes in tRNA modifications in Shewanella sp. 4 were investigated, along with using a more defined growth media and comparing it to previously conducted characterization. A multitude of methods were applied, such as transmission electron microscopy and mass spectrometry, to observe both the infection mechanisms and changes in tRNA modifications over the course of the infection. I found that the infection cycle of the phage-host pair is predictable and consistent with previously conducted research, lasting 3 hours until cell lysis. Plaque assay and SDS-PAGE showed the release of virions 2-3 h post-infection (p.i.), and the production of viral proteins within cells starting from 100 min p.i. An intriguing periodic change in cell turbidity was also observed already before cell lysis. Furthermore, the tRNA modifications m1A, m5U, m6t6A, and Cm undergo statistically significant changes or display high variance during the course of the infection when comparing infected and uninfected cells. These may affect tRNA structural stability, translational accuracy, and cleavage in the host cell, showing possible importance during the infection. Understanding the fundamentals of the infection mechanisms involved in this bacterium-bacteriophage pair gives further insight into their role in the Baltic Sea ecosystem. This is especially relevant for establishing Shewanella as a potential laboratory model for studying molecular mechanisms that further cold-active metabolism.

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.

Though single-celled organisms lack the ability to maintain their internal temperature, prokaryotes can grow between temperatures of <0 °C and up to 100 °C. Because the macromolecules of cells may be too stable, or denatured and inactive at non-permissible temperatures, these organisms must be able to adapt their interior to cope. In E. coli, the DNA is maintained by DNA-binding proteins and topology enzymes like DNA gyrase, and these proteins have been tracked and observed in cells at permissible temperatures, though little work had been done to characterise the activity and motion of DNA loci, DNA gyrase and the chromosomal compaction at different temperatures. Here, I used super-resolution fluorescence microscopy to track and image DNA and DNA gyrase at a range of temperatures. It was shown that the time-dependence of the mean square displacement (MSD) of DNA Loci behaves subdiffusively (MSD ~ ta, a <1) and that this subdiffusion coefficient itself is dependent on temperature. Additionally, it appeared that the subdiffusive scaling factor a differs between two timescales (< 500 ms and 0.5 – 10 s). It was also shown that the proportion of bound to unbound DNA gyrase changes from being equally DNA-bound and unbound at 23 °C while mostly DNA-unbound at 30 °C and 37 °C, and being mostly DNA-bound at 42 °C. It was shown that the compaction of the E. coli nucleoid did not change massively between steady-state temperatures, but did increase in size upon a shift from 37 °C to 23 °C. The DNA gyrase population shifting from unbound to mostly bound at 42 °C was interpreted to be due to an increase in cellular ATP concentrations, while the increased binding at 23 °C was thought to be because of a lack of available ATP trapping gyrase on the DNA. These results were lightly correlated with the nucleoid’s compaction across temperatures, where an abundance of free, DNA-unbound gyrase coincided with an increase in relative nucleoid size. The subdiffusive scaling coefficients of DNA loci at each temperature was thought to represent the fluidisation of the nucleoid and cytoplasm at 42 °C, but brings into question how the cell can maintain a similar flexibility and mobility of DNA between cold and warm temperatures.