Browsing by study line "Biokemi och strukturbiologi"

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.

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.

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.