Master theses submitted in 2016
Altanchimeg Altankhuyag (Aurelia Lewis, Vandana Ardawatia, Thomas Karlsson)
Phosphoinositide-binding motifs influence the nucleolar localization of ErbB3-binding protein 1 (EBP1)
Phosphoinositides (PIs) are glycerol-based phospholipids made of two fatty acids and an inositol ring which can be phosphorylated on 3 hydroxyl groups. PIs are important signaling molecules involved in numerous functions at the plasma membrane and other organelle membranes. In addition, PIs are present in the nucleus where they participate in nuclear processes, such as chromatin remodeling, transcription and mRNA processing. In a previous interactomics study, our lab aimed to gain further understanding of nuclear PI functions, and several nuclear proteins were identified as possible phosphatidylinositol(4,5) bisphosphate (PtdIns(4,5)P2)-binding proteins.
Amongst these proteins, ErbB3 binding protein 1 (EBP1) was identified. EBP1 is a ubiquitous and conserved protein, located in both the cytoplasm and nucleolus, and associated with cell proliferation and survival. In a previous study in our lab, EBP1 was shown to bind directly to several PIs via two different PI-binding sites consisting of two lysine rich PI-binding K/R motifs. These motifs are located at the N-terminal (65-KKEKEMKK-72) and at the C-terminal (364-RKTQKKKKKK-373) of the protein and the bold lysines were shown to be required for PI interaction. In this study, using interaction mutants with K to A mutations, we show that the C-terminal PI-bindingmotif contributes the most to the localization of EBP1 in the nucleolus.
In addition, three mutations found in the C-terminal motif in tumor samples, also affected the sub-localization of EBP1, suggesting an important role for EBP1 in the nucleolus in tumorigenesis. This study also revealed the presence of the class I PI3K catalytic subunit p110β and its product phosphatidylinositol(3,4,5) triphosphate (PtdIns(3,4,5)P3) together with EBP1 in the nucleolus. Taken together, these results show that EBP1 interacts directly with PIs and associate with PtdIns(3,4,5)P3 in the nucleolus. The presence of p110β and PtdIns(3,4,5)P3 in the nucleolus indicate their potential role in regulating nucleolar processes, at least via EBP1.
Syed Noor Ul Abideen
Syed Noor Ul Abideen (Rune Male)
Impact of potential drugs on the ecdysone receptor and molting of Lepeophtheirus salmonis larvae
Crustaceans are morphologically diverse organisms found in fresh and salt water and in terrestrial ecosystems. Some parasitic crustaceans are Rhizocephala, tongue worms and sea lice. Lepeophtheirus Salmonis is an ectoparasitic crustacean that feeds on salmon and trout inducing annual losses to salmon farming industry is several hundred millions US dollars. Salmon lice have become resistant to many of the available chemicals for use against the parasite, hence there is a strong need to develop new treatment methods to control L.salmonis including by interfering with nuclear receptors. Physiological events like growth, reproduction and molting are controlled by nuclear receptors which are ligand activated transcription factors, which could be good targets for drug design and to inhibit the molting of L.salmonis. Two such ligand activated transcription factors; ecdysone receptor (ECR) and ultraspiracle (USP), form a heterodimer complex that have a major role in the ecdysteroid signaling pathway. A third receptor in arthropods, Hormone receptor 38 (HR38) is also involved in regulating the ecdysteroid action possibly by competing with ECR on binding with USP. In vitro, testing of possible ligands were done with the use of Cercopithecus aethiops monkey kidneys (CV-1) cells, chimeric constructs of ECR-ligand binding domain (ECR-LBD) and USP-ligand binding domain +DNA binding domain (DBD) fused to yeast GAL4-DNA binding domain (DBD) and VP16 activation domain (AD) respectively. Cells were co-transfected with a luciferase reporter for the quantification of protein-protein interaction in a mammalian two hybrid system.
Ponasterone A (PonA) had high affinity compared to 20-hydroxyecdysone (20E), methyl farnesoate, juvenile hormone and 9-cis retinoic acid for the ECR/USP heterodimer complex. Assay of ecdysone agonist’s diofenolan, pyriproxyfen and fenoxycarb induced some but weak luciferase activity at higher concentrations and tebufenozide at lower concentrations. Chemicals/ligands like tetrahydroqunoline, methoprene, methoxyfenozide, juvenoid, dicyclanil, cyromazine and halofenozide had no such effects on the luciferase activity of reporter gene. Modulation of luciferase activity by co-treatment with pyriproxyfen, diofenolan, dicyclanil and tebufenozide combined with low concentration of PonA seemed to inhibit the actual activity of the reporter. Similar chimeric constructs as mentioned above were used for the USP and HR38 heterodimerization as well. Adding fenoxycarb as a ligand to cells expressing the heterodimer had no effects on the luciferase activity of reporter gene and confirmed that these receptors probably 2
are orphan receptors in L.salmonis alike in other arthropods. In vivo studies were conducted on the L.salmonis nauplia I larvae, testing effects of chemicals on the normal molting and morphology of L.salmonis nauplia II and copepods larval stages were documented. Chemicals like diacyclanil, fenoxycarb, diofenolan, methoprene, tebufenozide and PonA, each at 10 μM affected the normal molting of L.salmonis larvae. In conclusion, in vivo assay of nauplia I larvae of L.salmonis and in vitro cell based reporter assay on the ECR/USP heterodimerization in CV-1 cells with various agonists represents a powerful tool for screening endocrine disturbing chemicals (EDCs) against L.salmonis.
Essa Ahsan Khan
Essa Ahsan Khan (Kari Fladmark, Amanda Edson)
Establishment of transgenic zebrafish lines to detect oxidative stress-induced insults and to elucidate cellular stress protection ability of the Parkinsons disease-related protein DJ-1
Reactive oxygen species (ROS) are the products of incomplete reduction of molecular oxygen. Increased levels of ROS have been linked to cancer and neurodegenerative diseases. Among ROS, hydrogen peroxide (H2O2) and superoxide radical O2- are the most researched in biological systems because they are produced as side-products by a wide variety of enzymes. DJ-1 is an important cellular protein conferring protection against oxidative stress possibly by harnessing the properties of conserved a Cys106 residue. To model the importance of this residue, we aimed to establish transgenic lines of zebrafish with cell specific over-expression of zDJ-1C106A. Additionally, we also wanted to establish a transgenic line expressing the genetically encoded fluorescent probe: HyPer, that allows monitoring the intracellular H2O2 levels with a high level of sensitivity and specificity. Transgenic zebrafish lines with HyPer and zDJ-1C106A expression were developed utilizing Tol2 transposable vector system. The cell specific expression of these genes was ensured through a Gal4/UAS system. The driver lines with GalFF expression in glial and neuronal cells were crossed with the established responder lines tg(UAS:eGFP-2A-Flag-zDJ-1C106A) and tg(UAS:HyPer). We were unsuccessful in obtaining positive F1 off-springs of the HyPer expressing line. The established lines with glial tg(gfap:GalFF/UAS:eGFP-2A-Flag-zDJ-1C106A) and neuronal tg(eno2:GalFF/UAS:eGFP-2A-Flag-zDJ-1C106A) over-expressions of mutant DJ-1 were exposed to oxidative stress. Contrary to expected results, both transgenic lines were resistant to oxidative stress induced insults as compared to wild type.
Nikolai Isak Honorat
Nikolai Isak Honorat (Thomas Arnesen, Adrian Drazic)
Investigation into putative human N-terminal acetyltransferases
Espen Bariås (Thomas Arnesen, Håvard Foyn og Line Myklebust)
In vitro studies of N-terminal acetyltransferase inhibitors
Siri Øfsthus Goksøyr
Siri Øfsthus Goksøyr (Thomas Arnesen, Sylvia Varland)
Characterization of a novel isoform of
human N-alpha acetyltransferase 30
Martha Eimstad Haugstøyl
Martha Eimstad Haugstøyl (Johan Fernø, Marianne Borlaug)
Transcriptional effects of bile acids in adipocytes
Over the years, it has been well established that the beneficial effects of bariatric surgery are not solely due to gastric restriction and malabsorption, but also improved metabolic and endocrine regulation. A wide range of data have shown that post-operative changes in the levels and composition of bile acids in the blood are correlated to the metabolic improvement after bariatric surgery. Particularly, emerging data have demonstrated indirect bile acid effects through activation of the specific bile acid receptors TGR5 and FXR. A previously performed microarray analysis in our group identified transcriptional changes involved in adipose tissue function in patients before and one year after bariatric surgery. However, the role of bile acids in these changes have not yet been established.
In the present study, direct transcriptional effects of bile acids were investigated in 3T3-L1 cells and human primary adipocytes. An optimized differentiation protocol was established and demonstrated to be highly relevant for the bile acids effects observed. The secondary bile acid lithocholic acid (LCA) showed strikingly repressing effects on adipocyte-related genes during 3T3-L1 differentiation. In particular, the expression levels of PPARγ2, the master regulator of adipogenesis, were significantly downregulated, whereas the specific bile acid receptor TGR5 was upregulated in response to LCA. Functional measurements supported the transcriptional effects observed, demonstrating reduced lipid accumulation and insulin-mediated glucose uptake in LCA-treated 3T3-L1 cells. Overall, these data suggest a general repressing effect of LCA on the ability of the 3T3-L1 pre-adipocytes to transform into fully mature adipocytes. Similar to LCA, the primary bile acid chenodeoxycholic acid (CDCA), which has been found significantly increased after bariatric surgery, repressed transcriptional activity of adipocyte-related genes. Of note, this occurred without concomitant upregulation of TGR5. Interestingly, and in contrast to LCA and CDCA, the conjugated bile acid GCDCA induced the expression levels of adipogenic genes. LCA increased mRNA levels of the inflammatory marker TNFα. Another marker for inflammation, IL6, was upregulated in response to LCA, but downregulated when treated with a higher LCA dose. By contrast, TNFα was downregulated in response to CDCA whereas IL6 expression remained unchanged.
In human primary adipocytes, the effects of LCA on mRNA levels of adipocyte markers were opposite to the effects in 3T3-L1 cells. The transcriptional effects of CDCA however, were comparable to the findings in 3T3-L1 cells, with downregulated levels of the same adipocyte X markers. TGR5 expression in human primary adipocytes was not changed in response to neither LCA nor CDCA.
In summary, this study demonstrates direct transcriptional effects of different bile acids on markers for adipocyte function in 3T3-L1 cells and human primary adipocytes. These findings provide new insight that may be relevant for metabolic improvement associated with changes in bile acid serum concentrations after bariatric surgery.
Nomana Iqbal (Rune Male, Christiane Eichner)
Characterization of the dopamine receptor genes in salmon louse,
The Salmon louse is a parasite that has a direct and huge bearing on the economy of the Fisheries industry and survival of wild salmon and trout. It is a major threat for Salmonid population in the Northern Hemisphere, particularly in countries like Norway, Scotland, Ireland and Canada and to the aquaculture industry in Chile. The developing resistances against prevailing prophylactic strategies are increasingly becoming a problem for these nations that heavily depend on fishing and quaculture for its economy. There is an urgent need to address this issue by developing custom-built strategies to prevent sea-lice infestations.
Dopamine is an important chemical messenger that acts as a neurotransmitter, present in the central nervous system and periphery of both vertebrates and invertebrates. Dopamine receptors have been characterized in arthropods and they are important in regulating sexual function, neuronal development and feeding. In ticks dopamine receptor of type D1 has been shown to be involved in salivary secretions which assist in feeding on the host and dopamine receptor acts over two independent signaling pathways. To explore the role of dopamine receptors in L. salmonis, RNA interference studies were carried out. Knockdown of LsDopamine1 was significant but no effect on lice morphology was observed, whereas LsDopamine2 seems to exhibit a changed morphology to some extent. Sequence analysis, structure prediction and phylogeny for two dopamine receptor genes (LsDopamine1 and LsDopamine2) from the salmon louse genome showed that they belong to the family of rhodopsin-like GPCRs, seven-trans membrane spanning domains and show high sequence similarities to the dopamine receptors found in arthropods. Ontogenic expression analysis revealed that LsDopamine1 and 2 are expressed in adult male and copepodids respectively. In situ hybridization showed the presence of LsDopamine1 and 2 in subcuticular tissues for copepodids and in tegmental glands type 1 in preadult I female lice.
Silje Kjølle (Even Birkeland, Lars A. Akslen)
Mining the Hypoxic Breast Cancer Secretome for Potential Biomarkers
Although major advances in the understanding of cancer biology have been accomplished over the last two decades, breast cancer 5-year survival rates have been stable in this period. This demonstrates the need for new biomarkers for early detection and diagnosis, as well as prognosis and treatment. Hypoxia is an important driver of cancer progression, and rapidly growing tumors often result in intratumoral hypoxic regions. Hypoxia affects processes such as angiogenesis and metastasis, and in this way leads to tumor progression. Angiogenesis is mostly quiescent, but can be reactivated through increased secretion of proangiogenic proteins. The breast cancer secretome is therefore promising in the search for new biomarkers. The aims of this thesis are to identify differences in secretion of angiogenic proteins of normoxic cells and cells exposed to hypoxia, and differences in angiogenic potential between the luminal and basal-like breast cancer subtypes, in searching for new biomarkers in the breast cancer secretome.
Secreted proteins from the luminal cell lines BT474 and MCF7, and basal-like cell lines HS578 and MB231, were extracted from conditioned media, in normoxic (21 % O2) and hypoxic conditions (1.2 % O2) for 24 hours. Proteins were digested with trypsin in solution protein digestion and detected in label-free mass spectrometry analysis. The obtained data was analyzed using Perseus for bioinformatics, SPSS for statistics and FunRich for gene ontology analysis. Actin staining was done to see changes in cytoskeletal organization induced by hypoxia, proteins of interest related to angiogenesis were validated with ELISA, and HIF-1α accumulation was investigated with Western blot. A migration assay with endothelial HMVEC cells and conditioned media from basal-like cell lines was done to investigate potential induction of endothelial cell migration.
n total 1782 proteins were detected in the mass spectrometry analysis of conditioned media from normoxic and hypoxic conditions of the 4 breast cancer cell lines. In analysis of differences between subtypes in hypoxia, 255 proteins were significantly upregulated for the luminal cell lines, and 192 proteins for the basal-like cell lines. The analysis showed that the majority of proteins involved in angiogenesis were upregulated in basal-like compared to luminal cell lines. Comparing proteins III secreted from hypoxic and normoxic cells within each subtype resulted in 64 proteins with significantly increased secretion in hypoxia in luminal cell lines, and 66 proteins in basal-like cell lines, representing the proteins with hypoxia-induced secretion. Gene ontology analysis showed differences in cellular component and biological processes for proteins upregulated in hypoxia in luminal and basal-like subtypes, and that only 8 proteins were in common for the proteins upregulated in hypoxia for the subtypes. Statistical analysis of MS data and ELISA validation of VEGF, ANGPTL4, IL-6 and IL-8 showed that hypoxia induced secretion of VEGF from luminal cell lines and increased secretion from basal-like cell lines, ANGPTL4 had increased secretion from the more aggressive basal-like cell lines in addition to the luminal cell line MCF7 in response to hypoxia, and IL-6 and IL-8 were not affected by hypoxia but seen only secreted from basal-like cell lines.
The basal-like breast cancer cell lines have a stronger angiogenic signaling in response to hypoxia, compared to the luminal cell lines. The proteins seen significantly different are considered to be expressed from early response genes in relation to hypoxia. Hypoxia-induced VEGF secretion was shown, with higher secretion from basal-like cell lines. ANGPTL4 is one of the proteins found to be significantly upregulated in hypoxia for basal-like cell lines, and with no secretion in the less aggressive luminal cell line BT474. Hypoxia also induced secretion of several proteins involved in extracellular matrix degradation, migration and metastasis, including CTSB, which is one of 8 proteins significantly upregulated in hypoxia for both luminal and basal-like cell lines. Both ANGPTL4 and CTSB can be potential biomarkers for tumor progression in relation to hypoxia, and are potential targets for inhibition of hypoxia-related breast cancer metastasis.
Eline Ringnes Mejlænder-Larsen
Eline Ringnes Mejlænder-Larsen (Anders Molven, Randi Hovland)
Detection of Somatic Mutations in Pancreatic Ductal Adenocarcinomas: A Comparison of Sample Types and Analytical Methods
Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. This highly aggressive cancer form is often asymptomatic at early stages, which means that most patients are diagnosed at an advanced stage. Thus, only 20 % of those diagnosed with PDAC can undergo surgery, which is the only possible curative option. This means that there is an urgent need for more knowledge about the molecular genetics of pancreatic cancer with the aim of achieving earlier detection and diagnosis, as well as personalized treatment.
In this study, the goal was to compare different sample types and methods for somatic mutation detection in PDAC. Ninety percent of these tumors are found to harbor a KRAS mutation, which is why this gene was chosen for the initial analysis. We employed Sanger sequencing for KRAS analysis of DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue and both Sanger sequencing and PNA clamp assay for analysis of fresh-frozen tissue. With Sanger sequencing we observed KRAS exon 2 mutations in 12 of 14 cases for FFPE tissue and in 9 of 14 cases for fresh-frozen tissue. The more sensitive PNA clamp assay revealed one more positive case in fresh-frozen tissue. Deep sequencing using a panel covering 15 cancer- elated genes verified these findings in addition to detecting an exon 3 KRAS mutation in one case. Detection of tumor DNA in liquid biopsies appears a promising tool for early detection and we therefore analyzed the KRAS mutation status as a biomarker in plasma and pancreatic juice. Plasma was tested with the PNA clamp assay, but only 6 of the cases were possible to analyze, 3 of them being positive for a mutation (same as found in tumor tissue from the patient). All pancreatic juice samples were positive for a KRAS mutation in the PNA clamp assay. Some of the cases even exhibited several mutations. Most mutations detected with the PNA clamp assay were verified with both Sanger sequencing and deep sequencing. The latter also revealed two cases positive for an exon 3 KRAS mutation. Deep sequencing detected TP53 mutations in 6 tumor cases, all, except one, subsequently verified by Sanger sequencing. In addition to these, three TP53 mutations were found only in juice. Of the 13 other genes covered by the deep sequencing panel, only one mutation was detected in one case (FOXL2).
In conclusion, Sanger sequencing of DNA from FFPE tissue in PDAC appears to be very efficient when analyzing for somatic mutations in a limited gene region. Deep 2 sequencing is more time consuming, but when several gene regions are to be sequenced and many samples are to be processed, it is well suited for molecular genetic diagnostics of tumors. The selection of technique will also depend on the biological material available for analysis, and analysis of pancreatic juice may reveal interesting aspects of early cancer development.
Sina Rostami (Sarah Tete, Rebecca Cox, Åsne Jul-Larsen)
Hemagglutinin-specific antibody responses following 2009 pandemic H1N1 influenza vaccination in healthcare workers
Influenza, a respiratory virus, has lead to a substantial morbidity and mortality in humans. The most reliable way of preventing the infection is through vaccination. Due to the high rate of change in influenza surface glycoproteins, hemagglutinin HA) and neuraminidase (NA), vaccination does not provide broad and long-term immunity.
Generally, antibodies that are directed to the head domain of hemagglutinin are measured as surrogate correlates of protection in the hemagglutination inhibition (HI) assay. Seasonal influenza vaccines did not induce protection against the novel pandemic influenza H1N1 virus that started circulating in 2009 (A(H1N1)pdm09). A(H1N1)pdm09 vaccines were shown to induce HA stalk-specific antibodies that are broadly protective, in contrast to seasonal vaccines that induce strain-specific antibodies directed to the HA head domain and little or no HA stalk-specific antibodies. The purpose of this study was to evaluate in detail the HA head and stalk specific antibody responses to vaccination with AS03 adjuvanted A(H1N1)pdm09 vaccine Pandemrix® among 57 healthcare workers at Haukeland University Hospital (HUH) (Bergen, Norway). Serum samples were taken prior to and 21 days post vaccination. The antibody responses were studied using traditional assays, HI and microneutralization (MN), which measure HA receptor binding antibodies and neutralizing antibodies, respectively. The IgG avidity to the conserved HA stalk and the more variable HA head domains were assessed in avidity enzyme linked immunosorbent assay (avidity ELISA) assay. To dissect the IgG response towards the HA stalk and HA head domains, specific IgG1, IgG2, IgG3 and IgG4 subclass responses were quantified in ELISA. We also assessed the functional ability of the HA stalk antibodies to activate Natural Killer (NK) cells in antibody dependent cellular cytotoxicity (ADCC) assay. Based on HI titer prior to vaccination individuals were assigned to two groups based on whether they had seroprotective HI titer (HI titer ≥40). Group 1 (20 individuals) had baseline protective HI titer (HI titer ≥40) while group 2 (37 individuals) had baseline HI titer below the protective level. Consistent with the HI results, group 1 showed higher baseline MN titers and HA head and stalk specific IgG1 and IgG3 in ELISA. However, post-vaccination, the HI and MN titers increased did not differ significantly between the two groups. This was also the case with the concentration of IgG1 and IgG3. The IgG1 and IgG3 response was HA stalk dominant pre vaccination in both groups. However, post vaccination, the response remained HA stalk dominant for IgG3 but became HA head dominant for IgG1 in group 1. Of interest, the baseline avidity of HA head and HA stalk specific IgG was lower in group 1 compared to group 2. Furthermore, vaccination did not significantly increase the avidity of HA stalk and HA head specific antibodies. However, the stalk specific antibodies had higher avidity levels compared to the HA head specific antibodies at both time-points. In ADCC, NK cell activation was measured by the expression of CD107a and INF-γ in response to activation by HA stalk-specific antibodies. NK cell activation was not significantly different between the two groups, both at baseline and post vaccination levels. However, post vaccination, the percentage of NK cells expressing CD107a and INF-γ was significantly increased in both groups. This provides evidence that the HA stalk-specific antibodies are indeed available regardless of the extent to which the mainly neutralizing antibodies are present, but their importance might not be reflected in more conventional serological assays (i.e. HI).
The results of the present study have implications for vaccination strategies aimed at inducing higher levels of high avidity HA stalk-specific antibodies that may provide protection through non-neutralizing functions such as ADCC. These HA stalk-specific antibodies may provide a broader protection against different strains of seasonal and pandemic influenza viruses.
Parminder Kaur Bhambra
Parminder Kaur Bhambra (Thomas Arnesen, Håvard Foyn)
Investigating the biochemical activity of uncharacterized acetyltransferases
Lars J. Sverkeli
Lars J. Sverkeli (Mathias Ziegler)
Conversion of the rodenticide Vacor by enzymes of the NAD metabolism
Nicotinamide Adenine Dinucleotide (NAD) is an essential molecule for all living organisms. NAD has a wide variety of functions, both in energy metabolism and in signaling pathways, and is involved in key cellular functions including replication, gene silencing and DNA repair. Given the importance of NAD, the ability to control the cell’s NAD pool can be a very valuable asset from a therapeutic perspective. Recently, the NAD biosynthetic pathway has been extensively studied as a potential therapeutic target.
Vacor was a rat poison released in 1974, but was retracted from the market after several cases of Vacor poisonings causing diabetes and neuropathy, many of them fatal. Recently, it was demonstrated that Vacor is converted to a Vacor-derived analogue of Nicotinamide Mononucleotide (NMN), an intermediate of the NAD biosynthesis, by Nicotinamide Phosphoribosyl Transferase (NamPRT). This conversion was also found to be necessary for Vacor-induced cell death, linking Vacor toxicity to the NAD biosynthesis. This study aimed to further investigate whether Vacor, its nucleoside relative Vacor Riboside (VR) and the Vacor product Vacor Mononucleotide (VMN) are further converted by enzymes of the NAD metabolism, and how this might affect the cell. Additionally, the possible methylation of Vacor by Nicotinamide N-Methyl Transferase (NNMT) was investigated.
To investigate whether Vacor is methylated by NNMT, an expression vector was generated and transfected into HEK293 cells. By LC-MS analysis, it was determined that no methylated Vacor was produced, showing that Vacor is not a substrate for NNMT. To investigate the conversion of Vacor and its product by enzymes of the NAD biosynthetic pathway and its effect on the cell, HEK293- and HeLa cells were subjected to treatment with Vacor and VR. Cell viability was then assessed by resazurine-based viability assays and the cell’s content of NAD metabolites and Vacor derivatives was assessed using LC-MS. Vacor treatment was found to induce NAD depletion and cell death in HEK293 cells while HeLa cells were found to be resistant to Vacor. By LC-MS analysis, it was found that the Vacor product VMN is further converted to the NAD+ analogue VAD+, presumably by NMNAT2, a conversion that appears to be necessary for the cytotoxic effect of Vacor. VMN was also found in cells treated with VR and in-vitro analysis confirmed that both Nicotinamide Riboside (NRK) 1 and NRK2 can convert VR to product. However, no further conversion of VMN to VAD could be observed when cells were treated with VR. Consequently, VR was not found to have any detrimental effect on the cell with no observable decrease in viability or depletion of NAD upon VR treatment.
While the exact mechanism behind Vacor toxicity and Vacor-induced NAD depletion is still not known, this study provides valuable insight into the mechanism of Vacor toxicity and extends the link between Vacor and the NAD metabolism. With the NAD biosynthesis under scrutiny as a promising target for treatment of a number of diseases and Vacor emerging as a candidate drug interacting with the NAD biosynthesis, further investigating and understanding the mechanism behind Vacor toxicity is an interesting prospect for the future.
Joakim Brunet (Rune Male, Muhammad Tanveer Khan
Characterization of Doublesex and mab-3 related genes and a SoxE homolog in the salmon louse Lepeophtheirus salmonis
The salmon louse, Lepeophtheirus salmonis, is an ectoparasitic copepod that feeds of salmonids. Salmon lice are a health hazard to farmed and wild salmon populations, and is responsible for hundreds of millions of dollars in commercial losses for the industry. Treatment methods have been mainly chemical, resulting in the evolution of resistant salmon lice and a growing need for novel treatment methods. The foundation for such methods is knowledge about the salmon louse at the molecular level. Knowledge about sexual differentiation and development in the salmon louse is limited, but could prove invaluable if these processes can be manipulated. The Doublesex and mab-3 related (Dmrt) family of transcriptions factors are functionally conserved regulators of sexual development in metazoans. The Sox family of transcription factors are involved in a wide variety of developmental processes, including sexual development. Two Dmrt genes were found in the salmon louse genome, Dmrt1 and Dmrt2, and was together with a SoxE homolog characterized in order to elucidate basic mechanisms of sex determination and differentiation. Another purpose of the study was to find a sex-specific genetic marker, which could open the door for further research into the sexual development of the salmon louse. The two Dmrt genes are very low expressed in the salmon louse, with the highest relative expression in the larval stages. Both Dmrt genes show sexually dimorphic expression in the gonads of adult individuals. In-situ hybridization of one Dmrt gene showed expression in specialized and uncharacterized cells in the copepodid stage. Dmrt1 show a 30 nucleotide 3`splice acceptor variation, resulting in two protein products with a ten amino acid difference at the nauplius II stage. Dmrt2 show potential splicing, and potential length variation in 3`untranslated region at the nauplius II stage. RNA interference of Dmrt transcripts were unsuccessful in producing sufficient knockdown, hence the function of these genes remain unknown. The SoxE homolog show potential sex-specific splicing. Further studies on these three genes should be encouraged in regards to sexual development.
Christophe Balin (Øyvind Halskau, Martin Jakubec)
Expression and purification of misfolding peptides from E. coli for spectroscopic characterization
Neurodegenerative diseases are mainly caused by a single genetic mutation which can be inherited or induced by different factors, with the most frequent being age. These genetic mu-tations result in an uncontrolled production of misfolding proteins which reveals to form neuro-toxic oligomers affecting severely the psychological and motor conditions of diagnosed pa-tients. The mechanisms of neurotoxicity of those misfolding proteins are still uncertain and, in order to elucidate them, recent studies are now focusing on the mechanisms linked to oligo-meric formation and the interactions between oligomers and the cell membrane. One of the main limitations is the need of high amounts of pure misfolding protein for spectroscopic char-acterization. Here, we present the design of expression vectors by PCR mutagenesis, in order to overexpress recombinant Aβ1-42 and α-syn fusion proteins into E.coli cells, and their sub-sequent purification, as well as the development of a method aiming to prevent and reverse the formation of oligomers in solution, noticed after the cleavage of Aβ1-42 fusion protein, for the recovery of monomeric state proteins at concentration in the range 1-3 mg per liter of cul-ture. Eventually the study presents 2D-NMR characterization of the two targeted misfolding proteins at low concentrations by 1H-15N SOFAST-HMQC NMR. Future perspectives include extensive NMR characterization of the purified samples as well the formation of multiple lipid-protein nanodiscs models and their subsequent spectroscopic characterization by ThT assay, Circular Dichroism, Dyamic Light Scattering, and 2D/3D NMR.
Elise Moltzau Wanderås
Elise Moltzau Wanderås (Johan Fernø)
Establishment of multicolor flow cytometry to characterize myeloid immune cell composition in adipose tissue from obese patients
Obesity and type 2 diabetes (T2D) are recognized as chronic pro-inflammatory diseases.Macrophages, a type of innate immune cell, accumulate in adipose tissue (AT), where they are highly important mediators in adipose tissue inflammation. In metabolically healthy lean subjects, AT macrophages (ATM) macrophages fulfill their housekeeping functions including remodeling of the extracellular matrix and clearance of apoptotic cells. However, in obese subjects the macrophages change their phenotype and function as a cause of the proinflammatory environment. This change includes a so called polarization of the macrophages, which seems to be a key event in obesity-induced visceral adipose tissue (VAT) inflammation and development of insulin resistance. However, most studies on this issue come from model
organisms and the ATMs are poorly characterized in humans. The mechanism inducing the polarization from the anti-inflammatory to the pro-inflammatory phenotype still remains an open question. In the present study, multicolor flow cytometry and an optimized adipose tissue fractionation protocol were established. Fluorescent antibodies were titrated and an antibody panel was evaluated to be successful in macrophage characterization in human adipose tissue. Matched samples of peripheral blood, subcutaneous adipose tissue (SAT) and VAT were analyzed from each patient, where the ATMs CD11c+CD206+ and CD11c-CD206+ ATM populations were identified. In contrast and as expected, the CD11c+CD206- monocyte population could only be identified in blood. This suggests that monocytes change their phenotype when infiltrating adipose tissue. A previously performed phenotypic characterization in human AT have characterized two ATM subsets, the CD11c+CD206+ as the pro-inflammatory and CD11c- D206+ as the anti-inflammatory macrophage phenotype. By contrast, the data from this study suggests a similar phenotype for CD11c+CD206+ and CD11c-CD206+ subsets in both SAT and VAT. The similar phenotypic characteristic was observed between the two ATM populations in SAT and VAT when further characterization was made with CD44 and CD163, as they both expressed CD44, but lacked CD163 expression. Together, these findings provide new insight that may be relevant for future research on macrophage subsets and their phenotypic characteristics in adipose tissue.
Frida Thyri Segafredo
Frida Thyri Segafredo (Anna T. Wargelius, Lene Kleppe)
Exploring candidates for sterility in juvenile Atlantic Salmon (Salmo salar)
The Norwegian aquaculture is ever growing, and contributes to the economic growth in Norway. However, some challenges remain to be solved in order for the industry to further increase the production. One of the more important issues is the escape of farmed salmon from open water facilities into the natural environments of wild salmon, and the genetic introgression of farmed fish into wild populations (Glover, Quintela et al. 2012). Farmed salmon is also prone to early maturation, which is known to reduce growth rates, increase prevalence of disease and compromise animal welfare (Taranger, Carrillo et al. 2010). One approach to solve both these problems may be the use of sterile fish. Currently induction of triploidy is the only commercial solution to induce sterility in salmon. However, these fish are only suitable for cultivation in Northern Norway due to their sensitivity to higher water temperatures. Additionally, triploid fish are more prone to develop skeletal deformities, and the harvest at a whole generally has a lower quality score. Consequently, welfare considerations should be taken, and there is a need to develop alternative sterility methods.
Alternative sterility methods may target proteins essential for the development of primordial germ cells (PGC) (Nagasawa, Fernandes et al. 2013, Kleppe, Wargelius et al. 2015). Another possibility may be to target proteins essential for juvenile survival of germ cells. In order to induce sterility at the juvenile stage, the cells mediating the survival and growth of PGC need to be targeted. The somatic gonad contains many cell types including the Sertoli cells in males and the granulosa cells in females, which are nurturing the germ cells and are therefore essential for germ cell survival.
In order to identify possible target genes that could eliminate Sertoli cells, the gonad tissue of germ cell-free (GCF) male salmon (Wargelius, Leininger et al. 2016) was used. The GCF male gonads are rich in Sertoli cells and lack the high number of germ cells present in a normal testis; this makes the GCF testis suitable for searching for genes expressed in somatic gonadal cells. The transcriptome of wild type (WT) and GCF gonad testes (unpublished data, Kleppe et al.) were compared to each other, and to all tissues that were transcriptome sequenced in the salmon genome project (Lien, Koop et al. 2016). This revealed two major candidate genes, inha and st8sia6, which were strongly indicated to be expressed only in the somatic gonad.
The primary aim of this thesis was to experimentally determine the localization of inha and st8sia6 in the salmon gonad of both sexes. The secondary objective was to experimentally 6 determine the specific expression of these genes in the gonad of the animal. The third objective was to functionally study these genes using gene editing, with an ultimate goal of determining if these factors could be appropriate sterility target genes in salmon.
The study showed for the first time in Atlantic salmon that inha is expressed in granulosa cells of females and Sertoli cells of the male fish. Spatial expression of inha could be detected in immature, pre-pubertal (females) and mature (males) gonads, with higher expression level in the mature gonads in comparison to the immature counterpart in both sexes. Expression level screen of multiple tissue types for inha, revealed a clear tissue specificity of its gene expression to gonadal tissues. Spatial expression analysis of st8sia6 revealed for the first time in any species that expression of this gene could be detected in Sertoli cells of mature males and granulosa cells of pre-pubertal females. However spatial expression could not be detected in immature gonads of either sex. Gene expression level analysis supported this finding in males where lower expression of st8sia6 was detected in immature male gonads. For st8sia6 a tissue specificity screen revealed significantly higher expression of this gene in the ovaries of Atlantic salmon, while expression could also be detected in testis, gill, gut and brain. For both genes attempts to knockout these genes failed, therefore we could not elucidate their possible functions and suitability as sterility targets in Atlantic salmon. However, their promising tissue specificity to either Sertoli or granulosa cells in male and female gonads respectively, suggests that further attempts should be made to elucidate their functions and suitability as sterility targets in Atlantic salmon.
Sofya Romanyuk (Anni Vedeler, Ann Kari Grindheim)
Annexin A7 and Annexin A11 as mRNA-binding Proteins
Annexins belong to a family of Ca2+-binding proteins that are distinguished by their specific and similar core structure. For a long time, it was believed that their functions were only associated with the Ca2+-dependent binding to phospholipids. However, Annexin A2 has recently been shown to bind mRNA and influence specific mRNA transport and translation. It has been speculated that other Annexins may also function in regulation of mRNA function. Annexins A7 and A11 were observed both in the nucleus and the cytoplasm of PC12 cells by confocal imaging. Moreover, Annexin A11 is present in the midbody of dividing PC12 cells. The nuclear localization of Annexin A7 and, but to a lesser degree, Annexin A11 was inversely proportional to tumorigenicity of the breast cancer cells MCF-10A, MCF-7 and MDA-MB-231. Annexin A7 is enriched in the cytosol, while Annexin A11 is enriched in the cytoskeletal fraction of PC12 cells. The high-molecular-mass forms of Annexins A11 in the cytosol are covalently conjugated to ubiquitin and/or SUMO1. Annexins A7 and A11 are associated with translationally inactive mRNP complexes of the cytoskeletal fraction of PC12 cells, binding directly to mRNAs. In vitro coupled transcription and translation reactions were performed in the nuclease-treated rabbit reticulocyte lysate. Low concentrations (100 nM) of Annexin A7 or Annexin A11 stimulated the translation of c-myc chimeric mRNAs consisting of the c-myc 5’- and 3’-UTRs and the coding region of the reporter protein Renilla luciferase. However, when this concentration was increased to ≈3 μM and above, translation was dramatically inhibited by more than 90 %. The modified chimeric c-myc mRNA lacking the Annexin A2 binding site (Δ196-330) in its 5’-UTR showed a similar inhibition in the presence of 3-10 μM of Annexins A7 or A11, although lower concentrations showed no increase in translation efficiency. Both Annexins A7 and A11 were also able to inhibit translation of Annexin A2. The mRNA binding properties of Annexins A7 and A11 were further analysed by in vitro RNA-protein binding assays. Annexins A7 and A11 bind to the 5’- and 3’-UTRs of c-myc RNA, but are not dependent on the Annexin A2 binding site (Δ196-330) in the 5’-UTR of c-myc RNA for binding, raising the possibility that there may be other sites involved. Thus, the interactions of different Annexins with specific RNAs invite for further research
Regine Åsen Jersin
Regine Åsen Jersin (Simon Dankel)
Novel functions of the amino acid transporter SLC7A10 in adipocytes
Excess body fat accumulation, particularly in the intra-abdominal region, increases the risk of common metabolic diseases, including type 2 diabetes, cardiovascular disease, and several forms of cancer. In a systematic screen of candidate genes in obesity, our group previously found increased SLC7A10 expression in subcutaneous adipose tissue after bariatric surgery which lead to profound weight loss. Further, human genetic association studies have revealed type 2 diabetes-associated variants that influence adipose SLC7A10 expression. SLC7A10, a neutral amino acid transporter, is found in the plasma membrane of adipocytes, but its role in adipocyte metabolism has not been established. In the present translational study, we wanted to obtain more knowledge of SLC7A10 expression and function in adipocytes, and to investigate the protein’s potential in protecting against obesity and metabolic syndrome.
Western immunoblot analysis confirmed increased Slc7a10 protein expression in mature mouse adipocytes, including in isolated mitochondria. Furthermore, this study verified that SLC7A10 transports neutral amino acids over the adipocyte plasma membrane, and indicated facilitation of glycine uptake and L-alanine efflux in both mouse and human adipocytes. SLC7A10-dependent effects on mitochondria were indicated by a gene ontology analysis performed for SLC7A10 co-expressed genes in isolated adipocytes of lean and obese patients, and a causal impact on mitochondrial respiration was demonstrated by changes in maximal respiration and spare respiratory capacity after experimental inhibition of SLC7A10. Finally, Slc7a10b knock-out zebrafish showed increased body weight and decreased adiponectin expression in adipose tissue, revealing novel mechanisms by which SLC7A10 may mediate disease-protective effects.
Our data support a mechanistic model where SLC7A10 prevents metabolic disease by enhancing adipocyte mitochondrial respiratory capacity and adiponectin expression, likely as a result of sustained glycine uptake.
Lorentze Hope Hornnes
Lorentze Hope Hornnes (Lise B Gundersen, Ingvild Aukrust og P R Njølstad)
Development of functional tools for the analysis of hepatocyte nuclear factor 4 alpha (HNF4A) gene variants associated with diabetes
Diabetes Mellitus is a metabolic disease, characterized by chronically elevated blood glucose. Classical diabetes has been divided into two classes, type 1 diabetes (T1D) and type 2 diabetes (T2D), although other more rare forms exist. Of these, Maturity-onset diabetes of the young (MODY) is the most frequent; a monogenic form of diabetes with autosomal inheritance, onset in adolescence (before 25 years of age) and severe beta-cell dysfunction. Hepatocyte nuclear factor-4 alpha (HNF-4A) is a transcription factor encoded by the HNF4Agene and plays a role in the regulation of glucose metabolism and insulin secretion in pancreatic beta-cells and gluconeogenesis in the liver. Variants in the HNF4A gene are most commonly associated with MODY, and in more rare cases with T2D and the kidney disorder, Fanconi syndrome. Lack of functional characterization of HNF4A variants results in a poor understanding of the molecular mechanisms behind different HNF4A associated phenotypes of diabetes. By developing new investigation tools to functionally characterize several HNF-4A variants, we could measure their transactivation in two different luciferase reporter assays, in transfected HeLa and HepG2 cells regulated by the HNF1A and G6Pase promoters, respectively. HNF- A variant protein levels and subcellular localization was also determined. Both reporter systems demonstrated increased levels of activity in variant p.R76W, associated with MODY, hyperinsulinemic hypoglycemia (HH) and Fanconi syndrome, while protein levels were normal (equal to WT). In contrast, the variants p.S78N and p.R127W demonstrated reduced levels of activity, while normal protein levels. Furthermore, the HNF4A variants did not affect the nuclear sub-cellular localization of the protein in transient transfected HeLa cells. Since the majority of the HNF-4A variants are located in the DNAbinding domain (DBD), their effect on transactivation may be due to altered DNA-binding to target genes. Thus, further development of a method for studying the DNA- binding and analysis by electrophoretic mobility shift assay (EMSA) analysis is needed for a more detailed characterization of the HNF4A variants and the molecular mechanism by which they cause diabetes and additional complications like Fanconi syndrome.
Anny Gravdal (K. Fjeld, B. B. Johansson, A. Molven, P. R. Njølstad)
Functional characterization of Carboxyl Ester Lipase variants causing diabetes and exocrine dysfunction
The digestive enzyme carboxyl ester lipase (CEL) is mainly expressed in the acinar cells of the pancreas. We have previously identified disease-causing mutations in a variable number of tandem repeats (VNTR) domain, localised in the last exon of the CEL gene. Single base deletions (DEL1 or DEL4) in the VNTR lead to frameshifts and a truncated C-terminal of the CEL protein. Patients with these mutations suffer from exocrine pancreatic dysfunction and diabetes. In addition, a copy number variant of the CEL gene (CEL-HYB) with only 3 VNTR repeats has shown to be a novel risk factor for chronic pancreatitis.
In this study, we wanted to further explore the role of the C-terminal domain of CEL in human pancreatic disease. We aimed to investigate the functional properties of not only previously characterized pathogenic variants, but also CEL variants identified in healthy controls. We also wanted to investigate whether a C-terminal epitope-tag could have an impact on our CEL protein studies.
In HEK293 cells, we found that the expression and secretion of CEL variants varied with VNTR length and composition. Both pathogenic and normal CEL variants were observed in lysate and in insoluble fractions of the cell. However, secretion of the disease-causing variants was reduced compared to secretion of the normal protein. When comparing CEL variants with and without tag, we observed a higher amount of untagged proteins intracellularly. Additionally, we found that several CEL variants induced apoptosis in HEK293 cells, but unexpectedly, some of these variants also led to increased cell viability.
The CEL VNTR is known to be heavily O-glycosylated, which is thought to be important for proper folding, secretion and stability of the protein. Here, we obtained evidence that the three deletion variants (DEL1, DEL4 and DEL8), all with the same theoretical molecular mass (73 kDa) but different VNTR composition, are likely to contain different patterns of O-glycosylation. Furthermore, the variants differed in secretion with only the pathogenic proteins (DEL1, DEL4 and HYB) showing impaired secretion. We also confirmed that the pI increased from CEL-WT to CEL-DEL1 by isoelectric focusing (pI 5.1 and 9.5 respectively).
In summary, we have shown that the cellular fate of CEL protein variants is influenced by the C-terminal of the protein. In addition, we found that epitope-tags most probably increase the solubility and stability of CEL proteins HEK293 cells, which may have led to an underestimation of the pathogenic effect of CEL-DEL1, CEL-DEL4 and CEL-HYB shown in previous studies.
Lene Reed Hjorteset
Lene Reed Hjorteset (Øyvind halskau, Øyvind Strømland)
Molecular Characteristics of Peptide-Fatty Acid Complex Relevant for Tumoricidal HAMLET
The protein-fatty acid complex HAMLET (human α-lactalbumin made lethal to tumor cells) has been extensively studied for its specific toxicity towards tumors, its pharmacological potential and its biophysical properties. An important research goal has been the structural characterization of the complex. However, well-resolved structural characterization of HAMLET and other, similar protein-fatty acid (PFA) complexes has been difficult to achieve. Results from experiments have been conflicting and challenging to interpret, misinterpretations and uncertainties regarding properties of the complex has therefore slowed progress.
In the current work, we approach this problem by working with a simplified polypeptide component. Peptides designated A-Cage-C and A-Link-C are used to try to elucidate the conformation of a complex similar to HAMLET, by sing high-resolution spectroscopy. The peptides are derived from bovine α- lactalbumin (BLA), the bovine counterpart of the protein in HAMLET, and by using smaller part of the protein as a model system we hopefully overcome some of the difficulties met when studying the whole complex. A-Cage-C and A-Link-C consist of the helixes in BLA responsible for interacting with membranes and membrane permabilization, one of possible mechanism for cytotoxicity reported for BAMLET (Rammer, Groth-Pedersen et al., 2010).These motifs are also involved in forming complex with OA.
Both of the peptides was showed to form complexes with OA. This was seen by change in their elution profile on SEC, changes in Trp-fluorescence for A-Link- C, and in different NMR spectra set up to investigate the polypeptide and fatty acid component individually. Unfortunately, the complexes were not as easy to study with NMR as expected. The complexes are dynamic and heterogeneous with respect to sizes, and is therefore difficult to obtain clear enough NMR spectrums for assignation of amino acids and hence details about important amino acid in interactions with OA was not conclusive. Complexes were produced with an excess of OA, and the effect of removing this by SEC resulted in complexes more similar to the peptide alone. Both complexes induce leakage of liposomes, and this is an indicator of toxicity in the full-length complexes. Toxicity assays using flow cytometry and Presto Blue were performed, but unfortunately they gave no results.