Cellular responses to stress and kinase signaling activation : apoptosis and differentiation

Stressignaler avkänns många gånger av membranbundna proteiner som översätter signalerna till kemisk modifiering av molekyler, ofta proteinkinaser Dessa kinaser överför de avkodade budskapen till specifika transkriptionsfaktorer genom en kaskad av sekventiella fosforyleringshändelser, transkriptionsfaktorerna aktiverar i sin tur de gener som behövs för att reagera på stressen. En av de mest kända måltavlorna för stressignaler är transkriptionsfaktor AP-1 familjemedlemen c-Jun. I denna studie har jag identifierat den nukleolära proteinet AATF som en ny regulator av c-Jun-medierad transkriptionsaktivitet. Jag visar att stresstimuli inducerar omlokalisering av AATF vilket i sin tur leder till aktivering av c-Jun. Den AATF-medierad ökningen av c-Jun-aktiviteten leder till en betydande ökning av programmerad celldöd. Parallellt har jag vidarekarakteriserat Cdk5/p35 signaleringskomplexet som tidigare har identifierats i vårt laboratorium som en viktig faktor för myoblastdifferentiering. Jag identifierade den atypiska PKCξ som en uppströms regulator av Cdk5/p35-komplexet och visar att klyvning och aktivering av Cdk5 regulatorn p35 är av fysiologisk betydelse för differentieringsprocessen och beroende av PKCξ aktivitet. Jag visar att vid induktion av differentiering fosforylerar PKCξ p35 vilket leder till calpain-medierad klyvning av p35 och därmed ökning av Cdk5-aktiviteten. Denna avhandling ökar förståelsen för de regulatoriska mekanismer som styr c-Jun-transkriptionsaktiviteten och c-Jun beroende apoptos genom att identifiera AATF som en viktig faktor. Dessutom ger detta arbete nya insikter om funktionen av Cdk5/p35-komplexet under myoblastdifferentiering och identifierar PKCξ som en uppströms regulator av Cdk5 aktivitet och myoblast differentiering.

Viral and cellular modulation of virus-induced apoptosis in experimental infection models

The aim of this study was to investigate herpes simplex virus type 1 (HSV-1)- and measles virus (MV)-induced cell death. HSV-1 with deletion in genes encoding infected cell protein (ICP)4 and protein kinase Us3 (d120) induced apoptosis and cathepsin activation in epithelial (HEp-2) and monocytic (U937) cells. Inhibition of cathepsin activity decreased the amount of d120-induced apoptosis indicating that d120-induced apoptosis could be cathepsin-mediated. Also, HSV-1 infection increased caspase activation suggesting that d120-induced apoptosis is probably caspase-mediated. Cystatin treatment decreased the activity of cathepsins and the replication of HSV-1 indicating that cathepsins contribute to HSV-1 infection. Interestingly, d120 induced also necroptosis in monocytic cells. This is the first report on necroptosis in HSV-1- infected cells. MV induced apoptosis in uninfected bystander T lymphocytes, probably via interaction of MV-infected monocytes with uninfected lymphocytes. The expression of death receptor Fas was clearly increased on the surface of lymphocytes. The number of apoptotic cells and the activation of cathepsins and caspases were increased in MVinfected U937 cells suggesting that MV-induced apoptosis could be cathepsin- and caspase-mediated. Cystatin treatment inhibited cathepsin activities but not MV-induced apoptosis. Besides HSV-1-induced apoptosis, innate immune responses were studied in HSV-1-infection. HSV-1 viruses with either ICP4 and Us3, or Us3 deletion only, increased the expression of Toll-like receptor (TLR)3 and stimulated its downstream pathways leading to increased expression of type I interferon gene and to functional interferons. These findings suggest that besides controlling apoptosis, HSV-1 ICP4 and Us3 genes are involved in the control of TLR3 response in infected cell.

Novel Cellular Luminescence Probes for Immunological and Toxicological Assessments

Escherichia coli K-12 (pEGFPluxABCDEAmp) (E. coli-lux), constitutively emitting bioluminescence (BL), was constructed and its BL emitting properties tested in different growth and killing conditions. The BL emission directly correlated with the number of viable E. coli-lux cells, and when subjected to the antimicrobial agent, the diminishment of the BL signal was linked directly to the number of killed bacterial cells. The method provided a very convenient application, especially when compared to conventional plate counting assays. This novel real-time based method was utilized in both immunological and toxicological assessments. The parameters such as the activation phase, the lytic phase and the capacity of the killing of the serum complement system were specified not only in humans but also in other species. E. coli-lux was also successfully used to study the antimicrobial activities of insect haemolymph. The mechanisms of neutrophil activity, like that of a myeloperoxidase (MPO)-H2O2-halide system, were studied using the E. coli-lux approach. The fundamental role of MPO was challenged, since during the actual killing in described circumstances in phagolysosome the MPO system was inactivated and chlorination halted. The toxicological test system, assessing indoor air total toxicity, particularly suitable for suspected mold damages, was designed based on the E. coli-lux method. Susceptibility to the vast number of various toxins, both pure chemicals and dust samples from the buildings and extracts from molds, were investigated. The E. coli-lux application was found to possess high sensitivity and specificity attributes. Alongside the analysis system, the sampling kit for indoor dust was engineered based on the swipe stick and the container. The combination of practical specimen collector and convenient analysis system provided accurate toxic data from the dust sample within hours. Neutrophils are good indicators of the pathophysiological state of the individual, and they can be utilized as a toxicological probe due to their ability to emit chemiluminescence (CL). Neutrophils can either be used as probe cells, directly exposed to the agent studied, or they can act as indicators of the whole biological system exposed to the agent. Human neutrophils were exposed to the same toxins as tested with the E. coli-lux system and measured as luminol amplified CL emission. The influence of the toxins on the individuals was investigated by exposing rats with moniliniformin, the mycotoxin commonly present in Finnish grains. The activity of the rat neutrophils was found to decrease significantly during the 28 days of exposure.

Nanoclustering augmentation : a novel mechanism of Ras activation in cancer

Proteins of the Ras family are central regulators of crucial cellular processes, such as proliferation, differentiation and apoptosis. Their importance is emphasized in cancer, in which the isoforms H-ras, N-ras and K-ras are misregulated by mutations in approximately 20 – 30 % of cases. Thus, they represent major cancer oncogenes and one of the most important targets for cancer drug development. Ras proteins are small GTPases, which cycle between the GTP-bound active and GDP-bound inactive state. Despite the tremendous research conducted in the last three decades, many fundamental properties of Ras proteins remain poorly understood. For instance, although new concepts have recently emerged, the understanding of Ras behavior in its native environment, the membrane, is still largely missing. On the membrane Ras organizes into nanoscale clusters, also called nanoclusters. They differ between isoforms, but also between activation states of Ras. It is considered that nanoclusters represent the basic Ras signaling units. Recently, it was demonstrated that on the membrane Ras adopts distinct conformations, the so-called orientations, which are dependent on the Ras activations state. The membrane-orientation of H-ras is stabilized by the helix α4 and the C-terminal hypervariable region (hvr). The novel switch III region was proposed to be involved in mediating the change between different H-ras orientations. When the regions involved in this mechanism are mutated, H-ras activity is changed by an unknown mechanism. This thesis has explained the connection between the change of Ras orientation on the membrane and Ras activity. We demonstrated that H-ras orientation mutants exhibit altered diffusion properties on the membrane, which reflect the changes in their nanoclustering. The altered nanoclustering consequently rules the activity of the mutants. Moreover, we demonstrated that specific cancer-related mutations, affecting the switch III region of different Ras isoforms, exhibit increased nanoclustering, which consequently leads to stronger Ras signaling and tumorigenicity. Thus, we have discovered nanoclustering increase as a novel mechanism of Ras activity modulation in cancer. The molecular architecture of complexes formed on the membrane upon Ras activation is another poorly understood property of Ras. The following work has provided novel details on the regulation of Ras nanoclustering by a known H-ras-GTP nanoclustering stabilizer galectin-1 (Gal-1). Our study demonstrated that Gal-1 is not able to bind Ras directly, as it was previously proposed. Instead, its effect on H-ras-GTP nanoclustering is indirect, through binding of the effector proteins. Collectively, our findings represent valuable novel insights in the behavior of Ras, which will help the future research to eventually develop new strategies to successfully target Ras in cancer.

Monooxygenase Diversity and Oxygen Activation within Polyketide Antibiotic Biosynthesis

Molecular oxygen (O2) is a key component in cellular respiration and aerobic life. Through the redox potential of O2, the amount of free energy available to organisms that utilize it is greatly increased. Yet, due to the nature of the O2 electron configuration, it is non-reactive to most organic molecules in the ground state. For O2 to react with most organic compounds it must be activated. By activating O2, oxygenases can catalyze reactions involving oxygen incorporation into organic compounds. The oxygen activation mechanisms employed by many oxygenases to have been studied, and they often include transition metals and selected organic compounds. Despite the diversity of mechanisms for O2 activation explored in this thesis, all of the monooxygenases studied in the experimental part activate O2 through a transient carbanion intermediate. One of these enzymes is the small cofactorless monooxygenase SnoaB. Cofactorless monooxygenases are unusual oxygenases that require neither transition metals nor cofactors to activate oxygen. Based on our biochemical characterization and the crystal structure of this enzyme, the mechanism most likely employed by SnoaB relies on a carbanion intermediate to activate oxygen, which is consistent with the proposed substrate-assisted mechanism for this family of enzymes. From the studies conducted on the two-component system AlnT and AlnH, both the functions of the NADH-dependent flavin reductase, AlnH, and the reduced flavin dependent monooxygenase, AlnT, were confirmed. The unusual regiochemistry proposed for AlnT was also confirmed on the basis of the structure of a reaction product. The mechanism of AlnT, as with other flavin-dependent monooxygenases, is likely to involve a caged radical pair consisting of a superoxide anion and a neutral flavin radical formed from an initial carbanion intermediate. In the studies concerning the engineering of the S-adenosyl-L-methionine (SAM) dependent 4-O-methylase DnrK and the homologous atypical 10-hydroxylase RdmB, our data suggest that an initial decarboxylation of the substrate is catalyzed by both of these enzymes, which results in the generation of a carbanion intermediate. This intermediate is not essential for the 4-O-methylation reaction, but it is important for the 10-hydroxylation reaction, since it enables substrate-assisted activation of molecular oxygen involving a single electron transfer to O2 from a carbanion intermediate. The only role for SAM in the hydroxylation reaction is likely to be stabilization of the carbanion through the positive charge of the cofactor. Based on the DnrK variant crystal structure and the characterizations of several DnrK variants, the insertion of a single amino acid in DnrK (S297) is sufficient for gaining a hydroxylation function, which is likely caused by carbanion stabilization through active site solvent restriction. Despite large differences in the three-dimensional structures of the oxygenases and the potential for multiple oxygen activation mechanisms, all the enzymes in my studies rely on carbanion intermediates to activate oxygen from either flavins or their substrates. This thesis provides interesting examples of divergent evolution and the prevalence of carbanion intermediates within polyketide biosynthesis. This mechanism appears to be recurrent in aromatic polyketide biosynthesis and may reflect the acidic nature of these compounds, propensity towards hydrogen bonding and their ability to delocalize π-electrons.