Browsing by Subject "Biochemistry and Structural biology"

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  • Larkiala, Taru (Helsingin yliopisto, 2020)
    Calsyntenin-3 is a type I transmembrane protein, that is mainly expressed on the post-synaptic cell membranes. It belongs to the calsyntenin family that is part of the cadherin superfamily. Calsyntenin-3 consists of a cytosolic C-terminal region, a transmembrane domain and an extracellular N-terminal part, that consists of a laminin G-like domain (LNS) and two cadherin domains (CAD). Calsyntenin-3 is mainly expressed in the brain, but it can also be found in the heart, liver, pancreas, lung, skeletal muscle and placenta. Calsyntenin-3 has an effect on neurogenesis by affecting the development of excitatory and inhibitory synapses. It might also play a role in Alzheimer’s disease, as it has been found to be able to bind β-amyloid peptide, that is known to play a key role in the development of Alzheimer’s disease. Calsyntenin-3 acts as a synaptic adhesion protein, that binds to the post-synaptic neurexins with its extracellular region. However, the previous studies have contradicting results regarding the calsyntenin-3 domains that mediate the interaction between the calsyntenin-3 and neurexins. There is also disagreement whether calsyntenin-3 binds neurexin-α, neurexin-β or both. Because of these discrepancies, the aim of this master’s thesis study was to produce the calsyntenin-3 ectodomain constructs that contained either the two CAD domains, the LNS domain or all three domains, using baculovirus mediated protein production in insect cell cultures. These purified protein constructs were meant to be used for the determination of the binding domains. Unfortunately, only the purification of the calsyntenin-3 LNS domain was successful and the purification of the constructs, containing the CAD domains, was unsuccessful. A SEC-MALLS experiment, that was performed for the calsyntenin-3 LNS domain, revealed that it forms dimers in a solution, which is consistent with experiments performed with the LNS domain of human sex hormone‐binding globulin. The second aim of this master’s thesis study was to express the calsyntenin-3 ectodomain constructs on the surface of HEK293T cells and to test the binding between calsyntenin-3 and neurexins in a cell surface binding assay. The results of the cell surface binding assay indicated that the binding is mediated by the calsyntenin-3 CAD domains and that calsyntenin-3 binds to neurexin-α, but the binding to neurexin-β was not detected. However, the results from the cell surface binding assay were conflicting: the binding between the calsyntenin-3 full ectodomain construct and neurexin-α was not detected, but the binding was detected between calsyntenin-3 CAD ectodomain construct and neurexin-α. Therefore, the cell surface binding assay cannot be considered entirely reliable and should be repeated before making further conclusions.
  • Hakosalo, Vili (Helsingin yliopisto, 2021)
    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.
  • Silfvast, Josetta (Helsingin yliopisto, 2021)
    The signal recognition particle (SRP) targets newly synthesized secretory and membrane proteins from the cytosol to the translocon complex on the endoplasmic reticulum membrane. This highly specific co-translational protein targeting is essential for proteostasis by preventing the accumulation of proteins in the cytosol and the mistargeting of proteins. Defects in the SRP68 and SRP72 subunits of eukaryotic SRP have been linked to various inflammatory muscle diseases such as myopathy and myositis. The full role of these subunits in protein targeting and regulation of targeting is unknown. Previously the yeast SRP72 subunit has been degraded using an auxin-inducible degron (AID) system to explore the effect of depletion on protein targeting and cell viability, but the mammalian SRP72-AID has not yet been studied. The aim of this study was to deplete the mammalian SRP68 and SRP72 subunits using the AID system. This study revealed that in the case of SRP68-AID, approximately 65% of the protein is degraded after 2 hours. Respectively, 75% of SRP72-AID degrades after 2 hours and 85% after 4 hours. However, complete depletion of subunits was not achieved during 24 hours of auxin treatment. Quantification of depletion also showed that the strongest decrease in SRP occurs during the first 2 hours. This study demonstrated that mammalian SRP subunits can be depleted using the AID system, providing a good basis for further research to examine the effect of subunit depletion on protein targeting. This may help to solve the mechanisms of diseases associated with SRP68 and SRP72 defects and to develop therapeutics for them.
  • Taha, Lamia (Helsingin yliopisto, 2021)
    The endoplasmic reticulum (ER) is an important organelle of the cell where a high number of proteins are synthesized and modified to obtain their final structure. Therefore, the ER stress, which is caused by accumulation of unfolded proteins in the ER, is not to be taken lightly since it could contribute to many diseases, such as cancer and neurodegenerative diseases. The response to the ER stress is the unfolded protein response (UPR), which is an adaptive system that helps in adjusting for increased folding needs within the ER. One of the main protein branches in the UPR is inositol requiring enzyme 1 (IRE1). IRE1 detects the status of protein folding inside the ER and initiates the UPR signaling pathway to achieve either normal folding status or cell death. The aim of this research was to express yeast IRE1 in E.coli and human IRE1 in insect cells, purify with affinity chromatography and study the IRE1’s crystal structure with a small molecule modulator that could possibly enhance its activity. The protein was expressed successfully and purified with glutathione S-transferase (GST) tag, and the activity of the pure protein was determined. The structural studies were not fully completed since the absolute purity and yield that was necessary for crystallization was not achieved due to loss of protein during gel filtration and precipitation. Based on the results it is likely that the structure of the protein could be solved and further biochemical and structural studies with F10 are possible.
  • Salumäe, Astrid (Helsingin yliopisto, 2020)
    In biotechnological protein production and metabolic engineering, regulating the expression of genes is essential. For this, expression systems composed of promoters, terminators and transcription factors are essential. So far, majority of these systems use native promoters and transcription factors. That however rises two problems: 1) these systems usually work in only a set of closely related species, 2) native regulatory components can cause unintended expression levels due to the complexity of cellular regulation. Recently, a synthetic expression system (SES) was established for a wide range of fungal species. The transcription factor used in this system comprises an activation domain that originates from a virus. However, in the field of biotechnology and especially food industry, viral DNA constructs are not favorable because of customer concerns. In this paper, plant-derived activation domains were screened in Trichoderma reesei and Pichia pastoris using mCherry as a target gene for measuring the expression levels. The best expression systems were also tested for protein production in T. reesei and P. pastoris. We tested the production of two different proteins – a bacterial xylanase and a phytase. Two of the novel activation domains provided similar expression levels to the viral activation domain in both fungi. In addition, we developed optimized expression systems for an unconventional yeast from Zygosaccharomyces spp. using the novel transcription factors. The best SES version was used for secretion signal sequence screening for xylanase protein production. To further improve the use of T. reesei as a production host, the CRISPR-Cas9 system with the Cas9 D10A nickase version was tested for transformation of T. reesei. Here, we demonstrated the genomic integration and expression of Cas9 D10A nickase in T. reesei using the SES system with the novel plant-derived activation domain. Furthermore, we successfully transformed the T. reesei Cas9 D10A nickase expressing strain using only guide-RNAs and a donor DNA.
  • Ahlblad, Niklas (Helsingin yliopisto, 2021)
    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.