Master theses submitted in 2014

Øyvind Ødegård (R. Aasland, Ø. Halskau, C. Issalene, Ø. Strømland)

Non-conserved C-terminal regions in the histone recognition module CW, determines specificity, stability and affinity towards monomethylated histone tails

Histone modifications have an important role in epigenetic gene regulation and contribute to the cellular memory system. It has been well documented that certain patterns of histone modifications are associated with different states of gene expression. The mechanisms underlying these associations are, however, still poorly understood. Many nuclear proteins have histone recognition modules that can serve to recruit the proteins to relevant sites in chromatin. The CW domain is a family of histone recognition modules found in chromatin-related proteins. While all known CW domains have specificity for histone H3 methylated on lysine 4, different subfamilies of CW domains have different specificities for different methylation states. The C-terminal “lid regions" of the CW domains are highly variable and they have been proposed to contribute to the ligand specificity. To explore how the C-terminal "lid regions" contribute to ligand specificity, this project aims to measure the relative mobility of these regions in A. thaliana ASHH2 CW domain using NMR experiments. To support our claims, intrinsic tryptophan fluorescence thermal denaturation and ITC have been used. We have engineered three ASHH2 CW domain variants, which differ in length in their N- and C-terminal regions. We show that the lid regions are important for the stability of the domain, and that the protein adopts a more stable confirmation upon ligand binding. The C-terminal helix is mobile in the unbound state but becomes less mobile upon binding. We also show that K9 has a small effect on binding since doubly modified H3K4me1K9ac has a higher KD than H3K4me1. Furthermore, the implications for the binding mechanisms are discussed.

Kjetil Løtveit Thorstensen (S. Ellingsen, J. Rasinger)

Pesticides Induce Oxidative Stress in Zebrafish Embryo

Aquaculture is an industry in rapid global growth. The increased demand and therefore production of aquacultural goods is bound to drain natural marine resources if fish are to continue being fed feeds based solely on marine products. To compensate for the lack of traditional marine based feed, a mixture of agricultural and marine based feed can be used, replacing some of the fish oil with vegetable oil. However, the vegetable feed may include traces of pollutants such as pesticides commonly used in agriculture. Several pesticides are more toxic to marine animals than to terrestrial animals, and bioaccumulate to a higher degree in marine animals. Thus, introduction of pesticides through fish feed can have negative effects for both the fish and consumers of aquaculture products. Some of the most used pesticides, such as endosulfan (ESF) and chlorpyrifos (CPF) are neurotoxins designed to kill insects by interacting with their nervous system. Several pesticides also have a secondary toxic ability to induce reactive oxygen species (ROS), which can lead to oxidative stress in the fish.

To gain more knowledge about the ROS inducing effects of ESF and CPF, toxicology tests were done using embryos of zebrafish (Danio rerio) as a model system. From a 6 hours post fertilization (hpf) to 72 hpf dose response test, the lethal dosage was found to be 500,000 μg/L ESF and 20,000 μg/L CPF. A 14 days post fertilization survival test using the exposure time from 6 hpf – 72 hpf, indicated 50,000 μg/L ESF being a lethal concentration. ESF caused negative effects, such as malformations, late hatching and reduced activity in concentrations above 20,000 μg/L and all of these were dead by day 14. CPF exposure did not seem to affect development at concentrations up to 10,000 μg/L. H2DCFDA can be used to determine ROS generation in living cells. H2DCFDA freely diffuse into exposed cells and will produce an increasing green fluorescence signal with increasing levels of ROS in the cells. ESF caused an increasing fluorescence gradient from 10,000 μg/L to 50,000 μg/L. This increase was also seen in CPF, but the signal was much weaker and harder to differentiate. qPCR on isolated RNA from exposed embryos indicated that ESF had mostly a downregulatory effect on genes related to antioxidants and the gene cyp1a1, but might stimulate vtg1 expression at high levels. CPF had mostly stable expression, except for gclc, which had a significant upregulation. ESF induces more ROS than CPF at high concentrations, but CPF is more acutely lethal then ESF.

Kristin Gravdal (R. Male, L. Sandlund)

Interaction studies of Ecdysone receptor, Ultraspiracle and Hormone receptor-like 38 in the salmon louse (Lepeophtheirus salmonis)

The salmon louse, Lepeophtheirus salmonis, is an ectoparasitic crustacean harming wild and cultured salmonids and generating major financial losses in the salmon farming industry. As the salmon louse is becoming resistant to many of the chemical treatments currently used, the need for novel treatment methods is critical. Important physiological events regulated by nuclear receptors (NRs) such as reproduction, growth and moulting might be suitable targets for new pesticides, and commonly used insecticides targets and manipulates NRs involved in the moulting process. The NR superfamily consists of (mostly) ligand-dependent transcription factors that modulate genetic ‘programs’, typically affecting several hundred genes downstream. In arthropods, ecdysteroid-signalling pathways, mediated via NRs, are vital. Two NR proteins, Ecdysone receptor (EcR) and ultraspiracle (USP), make up the functional ecdysteroid heterodimer complex, through which ecdysteroid-signalling is mediated. In insects, ecdysteroid action is regulated by juvenile hormones (JHs), hypothesised to act via the binding of USP. Ecdysteroid action is further modulated by a third NR, hormone receptor-like 38 (HR38), by competing with EcR for USP-binding. HR38 is also speculated to bind EcR. Analysis of the amino acid sequences of LsEcR, USP and HR38 revealed that the ligand binding domain (LBD) was quite conserved in all three, although variations occurred. Since substitutions might affect local topography of the LBD, ligand-binding affinities might be affected contributing to the making of salmon louse-specific pesticides. To study interactions of the three L. salmonis NRs and their response to possible ligands, a mammalian two-hybrid system was used. Ponasterone A (PonA) was demonstrated to induce dimerization of EcR and USP in a dose-dependent relationship. 20-E, however did not affect dimerization. Two non-steroid compounds, JHIII and methyl farnesoate (MF) only marginally affected heterodimerization. However, a similar chemical, 9-cis retinoic acid might have a slight effect. HR38 interacted with USP and heterodimerization was not affected by any of the added compounds. EcR, however, did not seem to interact with HR38 in this assay. HR38 was demonstrated to reduce EcR/USP binding in one experiment, however no effect was found in a second experiment. The findings of this study contribute to the understanding of the ecdysteroid-signalling pathway in L. salmonis.

Mamata Khatri (R. Male, P. Bhattachan)

Characterization of Fushi Tarazu Factor 1 and Hormone like Receptor 39 in the salmon louse Lepeophtheirus salmonis

The sea lice, Lepeophtheirus salmonis, is an ectoparasite of Atlantic salmon, Salmo salar. Aquaculture industry of salmon fish, is currently facing major challenge to control the sea lice epizootic. Rapid development of resistance against most available treatments is a main concern; and has raised need of novel treatment methods. Nuclear receptors (NRs) are transcription factors involved in many physiological processes like reproduction, molting, maturation and metabolism. Some of these receptors are highly activated by binding to specific ligands or molecule. Their function can be influenced by designed molecules which can activate or inhibit and, hence seize vital processes. NRs, Fushi Tarazu Factor 1 (FTZ-F1) and Hormone like receptor 39 (HR39) are members of important signaling pathways mediated by ecdysone hormone. FTZ-F1 is reported to be a competence factor, with function in molting and also in regulating forthcoming ecdysteroid pulse, reproduction and viability and in some cases neuron development too. FTZ-F1 expression and subsequent repression of HR39 works in a crossregulatory network with some other NRs to modulate timing of several gene expression.

Deduced full length amino acid sequence of LsFTZ-F1 and LsHR39 revealed a conserve domains of NR. Both NRs encoded two variants, one short truncated (variant 1) and another full length (variant 2). Quantified relative expression level of LsFTZ-F1 and LsHR39 revealed constant expression with fluctuation at all parasitic developmental stages. Lowest expression throughout preadult stages in females, indicated sex determining function of FTZ-F1. Conversely, LsHR39 expression was higher in females in preadult 1 stage, but later expressed alike in both indicating role in male and female developmental processes. Both NRs were localized in most body part of the adult female lice, but slightly higher in ovary. Gradient of HR39 along maturation end of ovary, indicated possible role in egg development and synthesis process. To study the function, RNAi experiments in two separate developmental stages. FTZF1 knockdown in nauplius larva caused molting arrest, and both in nauplius and preadult female, knock-down effect was lethal suggest its importance in viability. No effects of HR39 in nauplius or in preadult female were observed. The attempt to examine opposing expression pattern of HR39 and FTZ-F1 in knockdown female adult lice by qPCR was not successful because, as no down-regulation was found compared to parallel control. Results in this study supports FTZ-F1 role in molting and survival of sea lice. However, any particular characteristic role of HR39 couldn’t be elucidated. More investigation is needed to verify these findings.

Morten Govasli Larsen (Ø. Halskau, Ø. Strømland)

Spectroscopic study of annular oligomer forming peptides derived from bovine α-lactalbumin

Amyloid proteins showing a high extent of structural plasticity are implicated in the cytotoxicity mechanisms in neurodegenerative diseases. Important afflictions include Alzheimer’s, Parkinson’s and Huntington’s disease as well as other amyloid diseases such as AL amyloidosis and prionic diseases such as Creutzfeldt–Jakob. All these diseases are linked to the misfolding of proteins into a specific form of aggregates composed of fibers termed amyloids and the formation of inclusion bodies.

These aggregates and inclusion bodies were long thought responsible for the observed toxicity of amyloid diseases. However, as the research effort has progressed the protein aggregates and inclusion bodies are now regarded to represent the end state of a series of events where toxicity is caused by earlier steps, and cytotoxicity is believed to manifest itself on the cells membranes.

The immature fibrils of several amyloid diseases alter the electrical activity and depolarize cell membranes. In addition, channel-like multimeric structures embedded in membranes have been observed for several amyloid proteins. In general the toxicity is believed to be due to influxes of Ca2+ trough these channels as toxicity is inhibited by known channel inhibitors or by removal of extracellular Ca2+. Thus, these channel-like structures might represent promising therapeutic targets.

Several protein-fatty acid complexes with the ability to kill a range of cancer cell lines while sparing differentiated, non-cancerous cells have been shown to share several features with amyloid proteins including conformational flexibility, interactions with membranes, formation of channel-like structures and the disruption of ionic homeostasis.

This work involved the study of two peptides designated A-Cage-C and A-Lnk-C, both derived from bovine α-lactalbumin which together with oleic acid forms the protein-fatty acid complex BLAOA. The parts of bovine α-lactalbumin implemented in these two peptides, designated helix A and C in the native protein, have been shown to interact with cell membranes, and alone the helixes induces leakage of LUVs and forms channel like structures on membranes.

During this work expression systems to allow expression of these peptides was constructed and A-Cage-C was characterized in terms of its ability to bind LUVs, to alter secondary structure when bound to membranes and to cause leakage of LUVs contents. In addition, high resolution NMR was employed in an attempt to solve the structures.

A-Cage-C was shown to bind to membranes, to cause leakage of the content of LUVs and to undergo secondary structure changes upon binding to LUVs. Its activity was for all experiments highest at pH values under its pI where A-Cage-C is positively charged suggesting a dependence of electrostatic interactions for its ability to interact with membranes. Using high resolution NMR we were able to assign half of the residues in A-Cage-C. Chemical shift indexing suggests that the helical content of Helix A and C are retained in the peptide for the sequence segments analysed.

Sandra Ninzima (T. Arnesen, C. Osberg)

Functional conservation of the N-terminal acetyltransferase Naa30 between yeast and humans

The majority of proteins in yeast and humans are subjected to Nα-terminal acetylation. This co-translational protein modification is catalyzed by one of six Nα-terminal acetyltransferases (NATs), NatA-NatF. The NATs are differentiated on the basis of their substrate specificity, determined by the N-terminal sequence of the substrate.

The NatC complex is composed of three subunits: Naa30 is the catalytic subunit, Naa35 is assumed to be a ribosome-anchoring auxiliary subunit and Naa38 is an auxiliary subunit of unknown function. The complex is conserved from yeast to humans and each of the three subunits is required for NatC-mediated N-terminal acetylation.

This study aimed to investigate the human NatC complex in yeast Saccharomyces cerevisiae. The yeast Golgi protein, Arl3, was used as a protein model due to its mislocalization to the cytosol in the absence of either the Naa30 or Naa35 subunit. Fluorescence microscopy of the Arl3 subcellular localization revealed that the human Naa30 could rescue the Arl3 localization phenotype in naa30 yeast cells but not in naa35 cells. These findings suggest a likely complex formation between human Naa30 and yeast Naa35 and hence a functional and structural conservation between the two Naa30 orthologous subunits. In addition, due to the lack of complementation in naa35 cells, human Naa30 is probably not capable of post-translational N-terminal acetylation of Arl3.

NatF/Naa60 is only found in higher eukaryotes and display overlapping substrate specificity with NatC. In addition, human Naa60 was recently found to localize to the organelles of the secretory pathway. Based on this, we expanded our study by including the human Naa60 in a similar phenotypic rescue of the Arl3 Golgi-localization. Our data indicate that human Naa60 is able to rescue the Arl3 localization phenotype in naa30cells and naa35 cells. These results strengthen the already established substrate redundancy between human NatC and Naa60. Further, we also propose that Naa60 may Nt-acetylate Arl3 in a post-translational manner at the Golgi.