Browsing by study line "Biokemi och strukturbiologi"

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.

The protein kinase C (PKC) enzyme is a type of peripheral membrane protein that is classified as a member of the Ser/Thr kinases superfamily. Its function is to add phosphate groups to serine or threonine residues in other proteins. The multitude of functions that PKC performs, and consequently its involvement in a diverse array of diseases, is attributed to the complicated nature of the enzyme: it consists of 12 distinct isoforms, each exhibiting minor variations in catalytic activity. Out of them, eight have therapeutic potential and utilize diacylglycerol (DAG) as a secondary signalling molecule. The research in this thesis is to perform computational modelling that assists an experimental research program searching for selective activators for PKC. The particular project involves combining molecular modelling with insight from structural biology. In concrete terms, three pieces of scientific work are involved: 1) The investigation of the behaviour in the membrane of drug candidate molecules that emerge as new scaffolds is developed by the experimental medicinal chemistry team. 2) Predicting the structure of PKCα, PKCδ, and PKCϵ using AlphaFold2 and evaluating their stability using molecular dynamic simulation. 3) Combining the predicted structures with DAG-containing membranes to examine predicted protein binding to the membrane. According to the results, 15 candidates out of 21 exhibited comparable behaviour to the positive control in terms of their angle of penetration into the membrane leaflets. Turning to regulatory domain prediction, all three proteins’ C1 and C2 domains were predicted with high confidence scores. All predicted structures showed root mean square deviation (RMSD) and root mean square fluctuation (RMSF) within the normal range, and most of their secondary structures were stable during simulation. When combined with the membrane, the C2 domains showed stable interactions with the membrane, and C2 binding to the membrane completely triggers C1 to bind to the membrane; however, there were some instabilities between the C1 domain binding and the membrane. This study highlights promising PKC activators, demonstrating the utility of computational modelling for identifying potential therapeutic agents.

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.

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.

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.