Molecular Biology Master theses submitted in 2017

Lena Elise Høyland, Mathias Ziegler, Magali R. van Linden

The manipulation of the peroxisomal NAD+ pool affects cellular functions and the mitochondrial NAD+ pool

NAD+ is a key regulator of cellular and organismal homeostasis involved in bioenergetic and signalling pathways. Most cellular NAD+-dependent processes take place in the nucleus, cytosol, mitochondria, and to a lesser extend in the peroxisomes. Apart from its importance for metabolic processes, the role of the peroxisomal NAD+ pool with regards to signalling and its relationship to other subcellular NAD+ pools remains unclear. In this study, the significance of the peroxisomal NAD+ pool was investigated by targeting the catalytic domain of poly-ADP-ribose polymerase 1 (PARP1) to the peroxisomes of 293 cells. Thereby, the peroxisomal NAD+ pool would be permanently diminished due to the continuous formation of poly-ADP-ribose.

The previously established stable 293 pexPARP1cd cell line was characterised with regards to the cellular and subcellular consequences of an increased peroxisomal NAD+ turnover. Stable expression of pexPARP1cd indeed lowered the cellular NAD+ and NADH content. Moreover, the polymers were steadily turned over possibly indicating the existence of peroxisomal NAD+-dependent signalling pathways. The enhanced peroxisomal NAD+ turnover did not affect cell proliferation. However, it resulted in several metabolic adjustments. The mRNA level of the peroxisomal NAD+ transport protein SLC25A17 was increased implying an increased peroxisomal NAD+ import. Furthermore, increased sensitivity to inhibition of the salvage pathway of NAD+ biosynthesis was observed. Since metabolism of reactive oxygen species is one of the most important peroxisomal functions, the effect of the stable expression of pexPARP1cd on peroxisomal ROS metabolism was assessed. Even though 293 pexPARP1cd cells exhibited an increased catalase activity and Catalase mRNA levels, no significant change in the cellular ROS levels was observed.

The increased peroxisomal NAD+ consumption caused by pexPARP1cd activity partially impaired peroxisomal fatty acid β-oxidation as indicated by the accumulation of fully saturated very long-chain fatty acid derivatives. This conclusion was further supported by the observation that the expression of the NAD+-dependent L- and D-bifunctional enzymes involved in peroxisomal β-oxidation was upregulated.Surprisingly, expression of the pexPARP1cd protein resulted in significantly decreased mitochondrial NAD+ levels indicating that the peroxisomal and the mitochondrial NAD+ pools are interconnected.

Collectively, the results of this study provide first evidence for important functional roles of the peroxisomal NAD+ pool. In the context of the recent identification of peroxisomal targets of ADP-ribosylation, it would appear that the significance of NAD+ metabolism in these organelles has so far been underestimated.

Rebecca Wangen, Frode Selheim

Optimization of Sample Preparation for Mass Spectrometry-Based Quantitative Analysis of All-Trans Retinoic Acid and Valproic Acid Treated Acute Myeloid Leukemia Patients

Acute Myeloid Leukemia (AML) is a type of blood cancer arising in the myeloid lineage of the hematopoietic progenitor cells. AML is the most common form of acute leukemia in adults and the incidence increases with advanced age. The highly heterogeneous biological variance in elderly patients makes it challenging to treat. To find new cancer biomarkers it is important to use optimized methods to be able to quantify the highest number of proteins and peptides as possible. In the present study, we investigated different methods for protocol optimization used for proteomic research. We also measured protein quantity of individuals in a specific AML patient cohort, to identify the effect of the All-trans Retinoic Acid (ATRA) in combination with Valproic Acid (VPA), looking for new possible cancer biomarkers.

The use of Sodium dodecyl sulphate (SDS) for protein extraction might cause interference with the LC-MS instrumentation. To avoid the risk of interference, we tested the use of sodium deoxycholate (SDC) in combination with Trifluoroacetic acid (TFA). The results revealed that we quantified approximately 3300 more peptides and 200 more proteins with the SDS containing protocol compared to the SDC TFA protocol when analyzing patient sample. Moreover, the storage conditions of AML cell lines frozen as a dried pellet and in 4% SDS was investigated and compared against the current use of 20% FBS/10% DMSO for biobanking storage buffer. The difference of quantification were found to be minor, albeit storage in 4% SDS allowed quantification of a slightly higher number of proteins and peptides, followed by storage as pellet and in 20% FBS/10% DMSO. More importantly, we found several proteins for GO terms involved in cell proliferation, cell death, regulation of phosphorylation and signal transduction underrepresented in 20% FBS/10% DMSO when compared to dried pellet and 4% SDS. Thus, caution must be practiced in data interpretation when using different protocols for preservation of AML samples. We also analyzed the effect of washing the cells with PBS for media removal. The results revealed a highly modified proteome and indicated cell burst, and were not used for further experiments.

Altogether, our results obtained from method optimization were used for the study of the ATRA+VPA cohort. Proteins related to the RA- and RX receptors as well as transcriptional repressor proteins that bind to methylated DNA were found to be higher expressed in the non-responding compared to the responding patients, including proteins for blocking of VII myeloid differentiation. Several up regulated histone proteins were found after two days ATRA+VPA treatment, but their impact can only be speculative, as this study was performed at the protein expression level. A further study, based on modifications and enrichment is needed to reveal the epigenetic modifications.

Tore-Andre Brodahl, Thomas Arnesen, Line Myklebust, Håvard Foyn

Development of novel N-terminal acetyltransferase inhibitors

Therese Rønvik Eidsnes, Kari Fladmark, Jon Vidar Helvik

The protective function of L-serine in a zebrafish model of β-methylamino-L-alanine-induced toxicity

The non-proteinogenic amino acid β-Methylamino-L-alanine (BMAA) has been found in various aquatic organisms, including species of cyanobacteria and diatoms. It has been under investigation as a possible causative factor of neurodegenerative disease, including Amyotrophic lateral sclerosis (ALS), Parkinson’s disease and Alzheimer’s. Its mode of action is postulated to be two-way: firstly as a glutamate receptor agonist and secondly through being misincorporated into proteins in place of L-serine. The latter possibly being the cause of the characteristic plaques of misfolded proteins observed in the relevant pathologies.

Our research group has established zebrafish as a model to investigate BMAA´s mode of action. Zebrafish has proven to be an excellent animal model for neurological research due to its significant similarities to the human neurological system, both in the peripheral neurons and in the overall organization of the brain.

The main focus of this study was to investigate the postulated misincorporation effect of BMAA. Both neuronal cell culture and zebrafish larvae were exposed to BMAA or co-exposed with L-serine. In the SH-SY5Y neuroblastoma cell line L-serine was shown to protect from BMAA-induced cell death. In zebrafish larvae L-serine was shown to protect from BMAA-induced down-regulation of heart rate. To enable detection of BMAA incorporation into endogenous proteins in zebrafish larvae we established a method for sample preparation for LC-MS/MS and optimized mass spectrometry detection of BMAA. Non-protein- and protein fractions from exposed zebrafish larvae were spiked with D3-labelled BMAA to enable LC-MS/MS-based quantitation of BMAA. Our data showed that L-serine significantly reduced the amount of BMAA detected in the protein-associated fraction, but had no significant effect on the amount of BMAA detected in the non-protein associated fraction. We further isolated neurons from BMAA-exposed transgenic larvae expressing fluorescently labelled neurons in order to identify a specific neuronal uptake of BMAA.

We have also shown that an inhibitor of fatty acid amide hydrolase (FAAH), a main regulator of the endocannabinoid system inhibited BMAA-induced down-regulation of heart rate and induction of oxidative stress in exposed larvae.

Silje Hjørnevik, Ingvild Aukrust, Janne Molnes, Pål Njølstad og Lise B. Gundersen

Unraveling the pathogenic effect of HNF1A gene codingvariants identified in a Danish cohort by functionalcharacterization

Maturity-Onset Diabetes of the Young (MODY) is a monogenic from of diabetes, characterized by autosomal dominant inheritance, severe pancreatic β-cell dysfunction and an early onset of disease (before around 35 years of age). Sequence variants in the hepatocyte nuclear factor-1 alpha (HNF1A) gene, encoding the transcription factor HNF-1A, are found to cause the most common form of MODY (MODY3) and have also been associated with the development of type 2 diabetes. Moreover, a substantial number of MODY cases are often misdiagnosed as type 1 or type 2 diabetes because of overlapping clinical features. In the present study, we investigated the functional consequence of ten HNF1A variants, identified by whole exome sequencing of individuals in a Danish cohort, to assess the pathogenic effect of these variants on HNF-1A protein function.

HNF1A cDNA variants were constructed by site-directed mutagenesis and functionally investigated in terms of transactivation activity, subcellular localization, protein level and DNA binding ability. Results revealed a significantly reduced transactivation potential for two variants. In addition, subcellular localization studies disclosed an impaired nuclear targeting for one of these variants. The establishment of fluorescent-based electrophoretic mobility shift assay (EMSA) made it possible to assess the DNA binding ability of the variants. In this assay some of the variants revealed a reduced ability to bind to a HNF-1A consensus sequence. Three protein variants are considered possibly pathogenic, as these demonstrated reduced results in some of the functional studies, albeit not to a significant degree. However, additional studies are warranted to confirm our findings for these three variants and elucidate their potential role in disease. The remaining variants investigated were associated with normal transcriptional activity and are thus considered non-causal.

Altogether, our study concludes two HNF1A variants to be pathogenic at a functional level, and thus may lead to MODY3 or susceptibility to type 2 diabetes by a loss-of-function mechanism; one by loss of DNA binding ability, impaired nuclear targeting and reduced transcriptional activity, and the other by a reduced ability to bind DNA and a reduction in transcriptional activity. The identification of pathogenic HNF1A variants has important implications for providing a proper diagnosis and a suitable treatment for the patients.

Nina Mc Tiernan, Thomas Arnesen, Line  Myklebust og Svein I. Støve

Functional characterisation of NAA10mutations potentially causing humandisease

N-terminal (Nt) acetylation is one of the most abundant covalent modifications ofproteins in eukaryotes (Aksnes et al., 2016). Approximately 80-90% of human proteinsare co- or post-translationally Nt-acetylated, a process which is catalysed by a group ofN-terminal acetyltransferases (NATs) (Arnesen et al., 2009). To date, six NATs (NatANatF)have been identified in humans all of which exert a distinct substrate specificity(Aksnes et al., 2016).

The most studied NAT, the NatA complex, is composed of the catalytic subunit Naa10and auxiliary subunit Naa15 (Arnesen et al., 2005a, Liszczak et al., 2013). In 2011, aNAA10 S37P point mutation was reported as the cause of a lethal X-linked disordernamed Ogden syndrome (Rope et al., 2011). This was the first described genetic disorder coupled to Nt-acetylation deficiency. Three years after the discovery of Ogdensyndrome, a novel NAA10 splice mutation was presented as the cause of Lenzmicrophtalmia syndrome (LMS) (Esmailpour et al., 2014). Since then, several other NAA10 mutations have been reported as cause of disease in patients with varyingdegrees of intellectual disabilities or developmental delays (Popp et al., 2014, Casey etal., 2015, Saunier et al., 2016).

This study undertakes the functional characterisation of five recently identified NAA10mutations which are suspected to be pathogenic in the patients harbouring these. These mutants include Naa10 I72T, E142K and K165R-frameshift (fs), which were identifiedin males, as well as Naa10 V111G and I99L, which were identified in females. The workpresented in this thesis demonstrates that Naa10 V111G is functionally impaired in itsmonomeric form both in vitro and in cellulo, while it maintains its functions as a part ofthe NatA complex. Naa10 I72T was shown to be affected similarly as Naa10 V111Gthrough in vitro investigations and previous work. Furthermore, Naa10 I99L was shownto impair NatA activity and have a mildly reduced catalytic activity in vitro. Although invitro investigations showed that Naa10 E142K was destabilised and catalyticallyimpaired and Naa10 K165R-fs seemed unaffected, further studies are needed for thesevariants in order to determine whether they are causative of disease.

Mona Synnøve Nilsen, Simon Dankel og Andre Greger Madsen

Adipocyte branched chain amino acid metabolism and differentiation capacity of different adipose tissue depots

Overweight and obesity associate with insulin resistance and increased risk of developing several diseases, where altered adipocyte mitochondrial function and lipid storage play a central role. Adipose tissue is an important complex organ linked to several functions in nutrient metabolism, and recent studies combining metabolomic and transcriptomic analyses have revealed a marked upregulation of genes involved in BCAA catabolism during adipogenesis, and raised BCAA levels observed in obese patients with insulin resistance have been linked to reduced BCAA catabolism in adipocytes.

The main objective of this study was to examine changes in BCAA metabolism during adipogenic differentiation by combining gene expression analysis with metabolomic analysis as well as other approaches. We wanted to evaluate if different adipose tissue depots show distinct BCAA metabolism, using an array of primary adipocyte cultures obtained from different depots of mice and some from humans, including white/beige and brown adipocytes.

Gene expression analysis of genes encoding enzymes in mitochondrial BCAA catabolism revealed a marked increase in mRNA levels during adipogenic differentiation of 3T3-L1 cells and in mature mouse and human adipocytes. Functional gene knock-down in 3T3-L1 cells and primary mouse white adipocyte cultures altered expression of genes in adipogenesis (Pparg2, Glut4), the TCA cycle (Sdhb) and lipogenesis (Acly, Acc1, Fasn). Due to large variability in lipid accumulation between the different analyzed primary adipocyte cultures, we could not draw any meaningful conclusions on differential BCAA metabolism between distinct adipose tissue depots. However, these cultures revealed a consistent relationship between certain BCAA metabolites and lipid accumulation.

Åsta Ottesen, Elisabeth O. Berge, Stian Knappskog og Reham Helwa

BRCA1 promoter methylation: the influence on gene expressionand the effect of long term drug treatment

Breast cancer is the most common type of cancer among woman all over the world, withover 1.67 million new cases in 2012. Heritable breast cancer is closely linked to mutationsin the tumor suppressor gene BRCA1, with up to 80% lifetime risk for developing breastcancer among women harboring a mutation in this gene. However, most breast cancer casesare sporadic and somatic mutations of the BRCA1 gene are rare. Furthermore, some tumorsshow BRCAness, despite being BRCA1 wild-type. Thus, it is of great interest to assessalternative mechanisms for inactivation of the BRCA1 gene, and addressing the missingcausality of many breast cancers. Furthermore, it is of great interest to assess themechanisms of drug resistance, a major challenge in cancer treatment today, where BRCA1may play an important role.

The overall aim of this thesis is to increase the understanding of the biological role ofBRCA1 promoter methylation in breast cancer. Three sub aims for the present project wereoutlined; 1) Quantify the BRCA1 a and b transcripts and the total BRCA1 protein levelsand relate the expression data to the methylation pattern in the BRCA1 promoter region ina panel of breast cancer cell lines. 2) Investigate how the total expression levels, as well asthe ratio between the a and b transcripts are affected by alterations in the a and b promoterregion of BRCA1, including methylation of specific CpGs as well as the polymorphismsrs71361504 and rs799905. 3) Investigate the effect of long term treatment with the drugsolaparib and doxorubicin on the BRCA1 promoter methylation in SKBR3 breast cancercells as a potential cause of drug resistance.

The study showed a weak correlation between BRCA1 methylation pattern and BRCA1mRNA expression. No correlation was observed between the methylation pattern andprotein expressed or between mRNA levels and protein expression. Analysis ofpolymorphisms rs71361504 and rs799905 found in the BRCA1 promoter showed that thetwo variants seemed to counter-balance each other, giving equal luciferase expressionlevels when differing in two positions and lower expression levels when intermediatevariants were studied. Finally, long term drug treatment of the cell line SKBR3 did notalter the methylation levels in the BRCA1 promoter, consequently demethylation seems notto be a mechanism for drug resistance in the experimental setup tested in this study.

Tonje Merethe Røssland, Thomas Arnesen, Henriette Aksnes og Michael Bruno

Investigation of Novel PotentialN-Terminal Acetyltransferase Enzymes

Ole Petter Nordbø, Simon Dankel og Jan Inge Bjune

A novel IRX5-APP pathway in adipocyte thermogenesis and obesity

Obesity (defined as BMI of >30 kg/m2) is an expanding worldwide problem and has serious consequences, leading to higher risk of developing diabetes, cardiovascular disease, cancer, neurodegenerative diseases and depression. Obesity-related diseases are thus the leading cause of death worldwide. If current trends continue, about 60% of the adult world population is projected to be obese by the 2030s.

The framework for this thesis was the finding that the iroquois transcription factor IRX5 inhibits inducible non-shivering thermogenesis, a process beneficial for metabolic health, in white/beige adipocytes. People who are homozygous for a high risk single nucleotide polymorphism (rs1421085) in the Fat mass and obesity related (FTO) are on average 3-4 kg heavier than carriers of the non-risk allele, and the polymorphism is very common in people suffering from extreme obesity. Although our group recently implicated IRX5 in mitochondrial uncoupling and "browning" of white adipose tissue, not much is known regarding the mechanism for IRX5 inhibition of adipocyte browning, including the specific genes under the control of IRX5. Further, there are no reports on a role for IRX5 in brown adipocytes. Elucidation of the functions of IRX5 in adipocytes may improve our understanding of the genetic makeup of obesity and poor metabolic health.

Through genome-wide analyses, our group recently identified the amyloid precursor protein (APP) as a putative IRX5 target gene (unpublished data). In this thesis, we investigated App regulation and metabolic effects following manipulation of Irx5 expression in cultured immortalized primary cells obtained from inguinal white, subscapular white and subscapular brown adipose tissue of mice, as well as in the browning-competent ("beige") mouse cell line ME3. This was achieved by transfection of small interference RNA (siRNA), as well as the gene editing method CRISPR-Cas9.

We detected IRX5 protein expression specifically in the nucleus of both white and brown adipocytes. Transient siRNA knockdown of Irx5 resulted in a marked increase in basal oxygen consumption rate and proton leak (thermogenesis) in brown adipocytes, based on real-time mitochondrial respiration measurements using the Seahorse XF instrument. siRNA-mediated knockdown of App mimicked these effects. Further, transient knockdown of Irx5 in brown adipocytes significantly decreased App mRNA levels, suggesting that App might mediate the inhibitory effects of Irx5 on adipocyte thermogenesis.

Further, we sought to establish stable monoallelic Irx5 knockdown clonal cell lines using CRISPR-Cas9. Comparing the stable knockdown cells to controls, no detectable reduction in Irx5 protein was seen in ME3 cells, while in primary inguinal cells there was a 50% reduction in Irx5 protein, corresponding to a marked reduction in App mRNA.

Our data show that IRX5 and APP inhibit basal oxygen consumption, mitochondrial uncoupling and thermogenesis in brown adipocytes, indicating that APP may at least partly mediate the IRX5-dependent effect of the FTO obesity risk locus.