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  • Pihlajamaa, Päivi (Helsingin yliopisto, 2014)
    Androgens are steroid hormones that regulate the development and function of male reproductive organs as well as physiology of many non-reproductive tissues, such as muscle, bone, liver, and kidney. Moreover, androgen signaling is involved in several pathological conditions, most common of which is prostate cancer. In its target cells, testosterone or its more potent metabolite 5α-dihydrotestosterone regulates cellular processes by modulating gene expression through the androgen receptor (AR). Ligand-activated AR translocates to nucleus and binds to specific DNA sequences, called androgen response elements (AREs), at the regulatory regions of its target genes. AR cistromes, i.e., global maps of genomic AR occupancy, comprise thousands of AR-binding events primarily located at distal enhancers. AR-binding sites are characterized by distinct histone modifications, and AR recruitment is primed by pioneer factors capable of binding to compact chromatin. AR interacts with a plethora of coregulatory proteins that modify the local chromatin environment and interact with basal transcription machinery. These elements create the complex cellular landscape for androgen action. The purpose of this thesis was to study molecular determinants of context-specific AR functions in vivo in murine androgen-responsive tissues. The advantage of in vivo studies is that unlike in cancer cell models androgen target cells reside within their physiological environment with an intact AR pathway. In the first part of this work, an androgen reporter mouse line with the luciferase gene under androgenic control was created. In this model, luciferase activity is a measure of AR function, and it can be used for assessing in vivo effects of chemical compounds on AR signaling. The androgen reporter mice were treated with genistein, a phytoestrogenic compound to which people consuming soy products are also exposed. Previous studies have implied that genistein plays a potential role in prostate cancer prevention. The results in this thesis work showed that genistein exhibits tissue-specific effects on AR signaling in vivo. Furthermore, genistein modulates endogenous AR-mediated gene expression in prostate, supporting its potentially beneficial role in prostate carcinogenesis. In the second part of this work, genomic AR occupancy was examined using chromatin immunoprecipitation (ChIP) coupled with massively parallel sequencing (ChIP-seq). Distinct AR cistromes were identified in three androgen-responsive tissues: prostate, kidney, and epididymis. AR-binding events associate with tissue-specific transcription programs responsible for distinct physiological functions of androgens in these tissues. The key finding in this work was that tissue-specific AR binding is directed by divergent pioneer factors, and that previously identified forkhead box protein A1 (FoxA1) is prostate-specific rather than general pioneer factor for AR. Two novel pioneer factors for AR were identified in this study hepatocyte nuclear factor 4 alpha (Hnf4α) in murine kidney and activating protein 2 alpha (AP-2α) in murine epididymis. ChIP-seq was also utilized to study in vivo role and characteristics of selective AREs cis-elements not bound by other steroid receptors. Transgenic SPARKI mice have the second zinc finger of the AR DNA-binding domain swapped with the respective part of glucocorticoid receptor, resulting in a chimeric AR unable to bind to selective AREs. A significant proportion of in vivo binding events of wild-type AR were not shared by SPARKI AR in prostate and epididymis, highlighting the importance of selective AREs in AR-specific functions in vivo. Differential receptor binding was also linked to differentially expressed genes in the epididymides of wild-type and SPARKI mice. De novo sequence analysis revealed that the selective AREs are characterized by decreased sequence conversation, indicating that, counter-intuitively, AR selectivity in vivo is achieved by relaxed rather than increased cis-element stringency. In conclusion, both the AREs and the collaborating proteins contribute to precise AR-regulated transcriptional outcome in the context of native chromatin and distinct tissues. Overall, the results clarified several molecular mechanisms employed by AR in vivo that can potentially contribute to the development of better treatments and diagnostic tools for hormone-dependent disorders in the future.
  • Thompson, James (Helsingin yliopisto, 2006)
    The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.
  • Sarin, Peter (2010)
    Double-stranded RNA (dsRNA) viruses encode only a single protein species that contains RNA-dependent RNA polymerase (RdRP) motifs. This protein is a central component in the life cycle of a dsRNA virus, carrying out both RNA transcription and replication. The architecture of viral RdRPs resembles that of a 'cupped right hand' with fingers, palm and thumb domains. Those applying de novo initiation have additional structural features, including a flexible C-terminal domain that constitutes the priming platform. Moreover, viral RdRPs must be able to interact with the incoming 3'-terminus of the template and position it so that a productive binary complex is formed. Bacteriophage phi6 of the Cystoviridae family is to date one of the best studied dsRNA viruses. The purified recombinant phi6 RdRP is highly active in vitro and possesses both RNA replication and transcription activities. The extensive biochemical observations and the atomic level crystal structure of the phi6 RdRP provides an excellent platform for in-depth studies of RNA replication in vitro. In this thesis, targeted structure-based mutagenesis, enzymatic assays and molecular mapping of phi6 RdRP and its RNA were used to elucidate the formation of productive RNA-polymerase binary complexes. The positively charged rim of the template tunnel was shown to have a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. This work demonstrated that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi6 RdRP can be greatly enhanced. Furthermore, proteolyzed phi6 RdRPs that possess a nick in the polypeptide chain at the hinge region, which is part of the extended loop, were better suited for catalysis at higher temperatures whilst favouring back-primed initiation. The clipped C-terminus remains associated with the main body of the polymerase and the hinge region, although structurally disordered, is involved in the control of C-terminal domain displacement. The accumulated knowhow on bacteriophage phi6 was utilized in the development of two technologies for the production of dsRNA: (i) an in vitro system that combines the T7 RNA polymerase and the phi6 RdRP to generate dsRNA molecules of practically unlimited length, and (ii) an in vivo RNA replication system based on restricted infection with phi6 polymerase complexes in bacterial cells to produce virtually unlimited amounts of dsRNA. The pools of small interfering RNAs derived from dsRNA produced by these systems were validated and shown to efficiently decrease the expression of both exogenous and endogenous targets.
  • Gylling, Annette (Helsingin yliopisto, 2008)
    Hereditary non-polyposis colorectal carcinoma (HNPCC; Lynch syndrome) is among the most common hereditary cancers in man and a model of cancers arising through deficient DNA mismatch repair (MMR). It is inherited in a dominant manner with predisposing germline mutations in the MMR genes, mainly MLH1, MSH2, MSH6 and PMS2. Both copies of the MMR gene need to be inactivated for cancer development. Since Lynch syndrome family members are born with one defective copy of one of the MMR genes in their germline, they only need to acquire a so called second hit to inactivate the MMR gene. Hence, they usually develop cancer at an early age. MMR gene inactivation leads to accumulation of mutations particularly in short repeat tracts, known as microsatellites, causing microsatellite instability (MSI). MSI is the hallmark of Lynch syndrome tumors, but is present in approximately 15% of sporadic tumors as well. There are several possible mechanisms of somatic inactivation (i.e. the second hit ) of MMR genes, for instance deletion of the wild-type copy, leading to loss of heterozygosity (LOH), methylation of promoter regions necessary for gene transcription, or mitotic recombination or gene conversion. In the Lynch syndrome tumors carrying germline mutations in the MMR gene, LOH was found to be the most frequent mechanism of somatic inactivation in the present study. We also studied MLH1/MSH2 deletion carriers and found that somatic mutations identical to the ones in the germline occurred frequently in colorectal cancers and were also present in extracolonic Lynch syndrome-associated tumors. Chromosome-specific marker analysis implied that gene conversion, rather than mitotic recombination or deletion of the respective gene locus accounted for wild-type inactivation. Lynch syndrome patients are predisposed to certain types of cancers, the most common ones being colorectal, endometrial and gastric cancer. Gastric cancer and uroepithelial tumors of bladder and ureter were observed to be true Lynch syndrome tumors with MMR deficiency as the driving force of tumorigenesis. Brain tumors and kidney carcinoma, on the other hand, were mostly MSS, implying the possibility of alternative routes of tumor development. These results present possible implications in clinical cancer surveillance. In about one-third of families suspected of Lynch syndrome, mutations in MMR genes are not found, and we therefore looked for alternative mechanisms of predisposition. According to our results, large genomic deletions, mainly in MSH2, and germline epimutations in MLH1, together explain a significant fraction of point mutation-negative families suspected of Lynch syndrome and are associated with characteristic clinical and family features. Our findings have important implications in the diagnosis and management of Lynch syndrome families.
  • Laurikkala, Johanna (Helsingin yliopisto, 2004)
  • Biala, Agnieszka (Helsingin yliopisto, 2011)
    Hypertension is a major risk factor for stroke, ischaemic heart disease, and the development of heart failure. Hypertension-induced heart failure is usually preceded by the development of left ventricular hypertrophy (LVH), which represents an adaptive and compensatory response to the increased cardiac workload. Biomechanical stress and neurohumoral activation are the most important triggers of pathologic hypertrophy and the transition of cardiac hypertrophy to heart failure. Non-clinical and clinical studies have also revealed derangements of energy metabolism in hypertensive heart failure. The goal of this study was to investigate in experimental models the molecular mechanisms and signalling pathways involved in hypertension-induced heart failure with special emphasis on local renin-angiotensin-aldosterone system (RAAS), cardiac metabolism, and calcium sensitizers, a novel class of inotropic agents used currently in the treatment of acute decompensated heart failure. Two different animal models of hypertensive heart failure were used in the present study, i.e. hypertensive and salt-sensitive Dahl/Rapp rats on a high salt diet (a salt-sensitive model of hypertensive heart failure) and double transgenic rats (dTGR) harboring human renin and human angiotensinogen genes (a transgenic model of hypertensive heart failure with increased local RAAS activity). The influence of angiotensin II (Ang II) on cardiac substrate utilization and cardiac metabolomic profile was investigated by using gas chromatography coupled to time-of-flight mass spectrometry to detect 247 intermediary metabolites. It was found that Ang II could alter cardiac metabolomics both in normotensive and hypertensive rats in an Ang II receptor type 1 (AT1)-dependent manner. A distinct substrate use from fatty acid oxidation towards glycolysis was found in dTGR. Altered cardiac substrate utilization in dTGR was associated with mitochondrial dysfunction. Cardiac expression of the redox-sensitive metabolic sensor sirtuin1 (SIRT1) was increased in dTGR. Resveratrol supplementation prevented cardiovascular mortality and ameliorated Ang II-induced cardiac remodeling in dTGR via blood pressure-dependent pathways and mechanisms linked to increased mitochondrial biogenesis. Resveratrol dose-dependently increased SIRT1 activity in vitro. Oral levosimendan treatment was also found to improve survival and systolic function in dTGR via blood pressure-independent mechanisms, and ameliorate Ang II-induced coronary and cardiomyocyte damage. Finally, using Dahl/Rapp rats it was demonstrated that oral levosimendan as well as the AT1 receptor antagonist valsartan improved survival and prevented cardiac remodeling. The beneficial effects of levosimendan were associated with improved diastolic function without significantly improved systolic changes. These positive effects were potentiated when the drug combination was administered. In conclusion, the present study points to an important role for local RAAS in the pathophysiology of hypertension-induced heart failure as well as its involvement as a regulator of cardiac substrate utilization and mitochondrial function. Our findings suggest a therapeutic role for natural polyphenol resveratrol and calcium sensitizer, levosimendan, and the novel drug combination of valsartan and levosimendan, in prevention of hypertension-induced heart failure. The present study also provides a better understanding of the pathophysiology of hypertension-induced heart failure, and may help identify potential targets for novel therapeutic interventions.
  • Lagus, Markus (Helsingin yliopisto, 2013)
    BACKGROUND Sleep disturbances and mood alterations are highly interrelated. The majority of patients suffering from depression report a reduced sleep quality. Inversely, people with sleep complaints are at elevated risk to develop depression. The complex regulation of these phenomena involves several brain areas and mechanisms. The susceptibility to change in this system is influenced by several factors, such as age and stressful lifestyle that are considered in this study. HYPOTHESIS The hypothesis of this study was that sleep and mood share common genetic/molecular regulatory networks and that both are also regulated by epigenetic mechanisms and neural plasticity. METHODS The studies were conducted both on humans and using an animal model for depression. In the animal model we measured the genome wide expression of genes in different brain areas of clomipramine-treated pups and adults. Using these data we conducted both individual area and inter-area network analyses of basal forebrain, frontal cortex, hypothalamus and hippocampus. We also measured the amount of BDNF, one of the plasticity-related factors, in sleep restriction and under aging. In the human study we conducted epigenetic analysis of the serotonin transporter gene and related the epigenetic changes to stress in a stressful working environment. RESULTS In the models investigated changes were observed on the system, protein, transcript and transcriptional regulatory levels. Inter-tissue pathways related to synaptic transmission, regulation of translation and ubiquitinylation were disrupted. The involved pathways are within the cellular components of the axons, growth cones, melanosomes and pigment granules. The disturbed networks are centred around serotonin, Mn(II) and Rhoa. In the basal forebrain the imbalance in gene expression is widely controlled by CREB1. Some of the changes seem to be epigenetically induced by sleep deprivation and stress. Individuals working in a high stress environment have significantly less methylation in the promoter area of serotonin transporter gene SLC6A4, as compared to individuals working in a low stress environment. We also found that the expression of cortical BDNF correlated with the recovery non-REM (NREM) slow wave activity (SWA) response, and that both the cortical BDNF and the SWA response to sleep deprivation were decreased in the aged animals, as were the changes in sleep latency. CONCLUSIONS The disturbances in the models investigated, arise, largely, but not solely, due to disruption in neurological systems previously related to the regulation of sleep and mood. Novelty value could be ascribed to findings that suggest involvement of inter-tissue networks, and more precisely, imbalance of melanosome related gene expression and gene networks connected to Mn(II). The stress induced demethylation of the SLC6A4 promoter suggests a mechanism for the body to cope with prolonged excessive stress. The downside of this coping mechanism is the possibility that this reprogramming increases the long-term risk for mood disorders. The findings in the sleep deprived aging rats support the hypothesis that the age related decrease in homeostatic NREM SWA is related to a reduced sleep need.
  • Wasik, Anita Agnieszka (Helsingin yliopisto, 2014)
    Diabetic nephropathy (DN) is a major microvascular complication of diabetes and a common cause of end-stage renal disease. Mechanisms leading to the development of DN are not fully understood, but podocyte injury is involved. Interestingly, in respect to glucose uptake podocytes are uniquely insulin sensitive cells. Insulin rapidly induces remodeling of the actin cytoskeleton and leads to glucose uptake via glucose transporters GLUT1 and GLUT4. Defects in the trafficking of the glucose transporters may affect the insulin sensitivity of podocytes. Thus, regulators of glucose transporter trafficking may provide suitable targets to enhance insulin sensitivity of podocytes and prevent the development and progression of DN. However, the precise mechanisms regulating glucose transporter trafficking and glucose uptake into podocytes are largely uncharacterized. To identify changes in the expression of glomerular proteins at an early stage of DN we performed quantitative proteomic profiling of glomeruli isolated from rats with streptozotocin-induced diabetes and controls. Ezrin was found to be downregulated in diabetic glomeruli. In cultured podocytes depletion of ezrin increased glucose uptake by increasing translocation of GLUT1 to the plasma membrane. Loss of ezrin also induced actin remodelling, which involved cofilin-1. Phosphorylated cofilin-1 was upregulated in diabetic glomeruli suggesting altered actin dynamics. Furthermore, reduced expression of ezrin was found in the podocytes of human patients with diabetes. We found that the filament-forming septin 7 forms a complex with CD2AP and nephrin, both of which are essential for glomerular ultrafiltration. We showed that septin 7 negatively regulates GLUT4 storage vesicle (GSV) trafficking by forming a physical barrier between the vesicles and the plasma membrane. The novel interaction partner of septin 7, nonmuscle myosin heavy chain IIA (NMHC-IIA), was found to positively regulate insulin-stimulated glucose uptake into podocytes. Loss of NMHC-IIA reduced formation of the SNARE complex involved in GSV exocytosis. Furthermore, we presented that insulin regulates the association of septin 7 and phosphorylated RLC (pp-RLC), a part of myosin hexameric complex, with a plasma membrane SNARE, SNAP23. pp-RLC is upregulated in diabetic glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with Type 1 diabetes. Our findings indicate that ezrin, septin 7 and NMHC-IIA regulate glucose uptake into podocytes and may play a role in the development of the renal complication in diabetes by regulating glucose transport and organization of the actin cytoskeleton in podocytes.
  • Nuotio, Krista (Helsingin yliopisto, 2007)
    Carotid artery disease is the most prevalent etiologic precursor of ischemic stroke, which is a major health hazard and the second most common cause of death in the world. If a patient presents with a symptomatic high-grade (>70%) stenosis in the internal carotid artery, the treatment of choice is carotid endarterectomy. However, the natural course of radiologically equivalent carotid lesions may be clinically quite diverse, and the reason for that is unknown. It would be of utmost importance to develop molecular markers that predict the symptomatic phenotype of an atherosclerotic carotid plaque (CP) and help to differentiate vulnerable lesions from stable ones. The aim of this study was to investigate the morphologic and molecular factors that associate with stroke-prone CPs. In addition to immunohistochemistry, DNA microarrays were utilized to identify molecular markers that would differentiate between symptomatic and asymptomatic CPs. Endothelial adhesion molecule expression (ICAM-1, VCAM-1, P-selectin, and E-selectin) did not differ between symptomatic and asymptomatic patients. Denudation of endothelial cells was associated with symptom-generating carotid lesions, but in studies on the mechanism of decay of endothelial cells, markers of apoptosis (TUNEL, activated caspase 3) were found to be decreased in the endothelium of symptomatic lesions. Furthermore, markers of endothelial apoptosis were directly associated with those of cell proliferation (Ki-67) in all plaques. FasL expression was significantly increased on the endothelium of symptomatic CPs. DNA microarray analysis revealed prominent induction of specific genes in symptomatic CPs, including those subserving iron and heme metabolism, namely HO-1, and hemoglobin scavenger receptor CD163. HO-1 and CD163 proteins were also increased in symptomatic CPs and associated with intraplaque iron deposits, which, however, did not correlate with symptom status itself. ADRP, the gene for adipophilin, was also overexpressed in symptomatic CPs. Adipophilin expression was markedly increased in ulcerated CPs and colocalized with extravasated red blood cells and cholesterol crystals. Taken together, the phenotypic characteristics and the numerous possible molecular mediators of the destabilization of carotid plaques provide potential platforms for future research. The denudation of the endothelial lining observed in symptomatic CPs may lead to direct thromboembolism and maintain harmful oxidative and inflammatory processes, predispose to plaque microhemorrhages, and contribute to lipid accumulation into the plaque, thereby making it vulnerable to rupture.
  • Vainio, Anni (Helsingin yliopisto, 2012)
    Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae are major health problems worldwide, both found in symptomless carriage but also causing even life-threatening infections. The aim of this thesis was to characterise MRSA and S. pneumoniae in detail by using several molecular typing methods for various epidemiological purposes: clonality analysis, epidemiological surveillance, outbreak investigation, and virulence factor analysis. The characteristics of MRSA isolates from the strain collection of the Finnish National Infectious Disease Register (NIDR) and pneumococcal isolates collected from military recruits and children with acute otitis media (AOM) were analysed using various typing techniques. Antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and the detection of Panton-Valentine leukocidin (PVL) genes were performed for MRSA isolates. Pneumococcal isolates were analysed using antimicrobial susceptibility testing, serotyping, MLST, and by detecting pilus islet 1 (PI-1) and 2 (PI-2) genes. Several international community- and hospital-associated MRSA clones were recognised in Finland. The genetic diversity among MRSA FIN-4 isolates and among FIN-16 isolates was low. Overall, MRSA blood isolates from 1997 to 2006 were genetically diverse. spa typing was found to be a highly discriminatory, rapid and accurate typing method and it also qualifies as the primary typing method in countries with a long history of PFGE-based MRSA strain nomenclature. However, additional typing by another method, e.g. PFGE, is needed in certain situations to be able to provide adequate discrimination for epidemiological surveillance and outbreak investigation. An outbreak of pneumonia was associated with one pneumococcal strain among military recruits, previously healthy young men living in a crowded setting. The pneumococcal carriage rate after the outbreak was found to be exceptionally high. PI-1 genes were detected at a rather low prevalence among pneumococcal isolates from children with AOM. However, the study demonstrated that PI-1 has existed among pneumococcal isolates prior to pneumococcal conjugate vaccine and the increased antimicrobial resistance era. Moreover, PI-1 was found to associate with the serotype rather than the genotype. This study adds to our understanding of the molecular epidemiology of MRSA strains in Finland and the importance of an appropriate genotyping method to be able to perform high-level laboratory-based surveillance of MRSA. Epidemiological and molecular analyses of S. pneumoniae add to our knowledge of the characteristics of pneumococcal strains in Finland.
  • Karkola, Sampo (Helsingin yliopisto, 2009)
    Breast cancer is the most common cancer in women in the western countries. Approximately two-thirds of breast cancer tumours are hormone dependent, requiring estrogens to grow. Estrogens are formed in the human body via a multistep route starting from cholesterol. The final steps in the biosynthesis include the CYP450 aromatase enzyme, converting the male hormones androgens (preferred substrate androstenedione ASD) into estrogens(estrone E1), and the 17beta-HSD1 enzyme, converting the biologically less active E1 into the active hormone 17beta-hydroxyestradiol E2. E2 is bound to the nuclear estrogen receptors causing a cascade of biochemical reactions leading to cell proliferation in normal tissue, and to tumour growth in cancer tissue. Aromatase and 17beta-HSD1 are expressed in or near the breast tumour, locally providing the tissue with estrogens. One approach in treating hormone dependent breast tumours is to block the local estrogen production by inhibiting these two enzymes. Aromatase inhibitors are already on the market in treating breast cancer, despite the lack of an experimentally solved structure. The structure of 17beta-HSD1, on the other hand, has been solved, but no commercial drugs have emerged from the drug discovery projects reported in the literature. Computer-assisted molecular modelling is an invaluable tool in modern drug design projects. Modelling techniques can be used to generate a model of the target protein and to design novel inhibitors for them even if the target protein structure is unknown. Molecular modelling has applications in predicting the activities of theoretical inhibitors and in finding possible active inhibitors from a compound database. Inhibitor binding at atomic level can also be studied with molecular modelling. To clarify the interactions between the aromatase enzyme and its substrate and inhibitors, we generated a homology model based on a mammalian CYP450 enzyme, rabbit progesterone 21-hydroxylase CYP2C5. The model was carefully validated using molecular dynamics simulations (MDS) with and without the natural substrate ASD. Binding orientation of the inhibitors was based on the hypothesis that the inhibitors coordinate to the heme iron, and were studied using MDS. The inhibitors were dietary phytoestrogens, which have been shown to reduce the risk for breast cancer. To further validate the model, the interactions of a commercial breast cancer drug were studied with MDS and ligand–protein docking. In the case of 17beta-HSD1, a 3D QSAR model was generated on the basis of MDS of an enzyme complex with active inhibitor and ligand–protein docking, employing a compound library synthesised in our laboratory. Furthermore, four pharmacophore hypotheses with and without a bound substrate or an inhibitor were developed and used in screening a commercial database of drug-like compounds. The homology model of aromatase showed stable behaviour in MDS and was capable of explaining most of the results from mutagenesis studies. We were able to identify the active site residues contributing to the inhibitor binding, and explain differences in coordination geometry corresponding to the inhibitory activity. Interactions between the inhibitors and aromatase were in agreement with the mutagenesis studies reported for aromatase. Simulations of 17beta-HSD1 with inhibitors revealed an inhibitor binding mode with hydrogen bond interactions previously not reported, and a hydrophobic pocket capable of accommodating a bulky side chain. Pharmacophore hypothesis generation, followed by virtual screening, was able to identify several compounds that can be used in lead compound generation. The visualisation of the interaction fields from the QSAR model and the pharmacophores provided us with novel ideas for inhibitor development in our drug discovery project.
  • Isaksson, Camilla Kristina (Helsingin yliopisto, 2007)
    The Parechoviruses (HPEV) belong to the family Picornaviridae of positive-stranded RNA viruses. Although the parechovirus genome shares the general properties of other picornaviruses, the genus has several unique features when compared to other family members. We found that HPEV1 attaches to αv integrins on the cell surface and is internalized through the clathrin-mediated endocytic pathway. During he course of the infection, the Golgi was found to disintegrate and the ER membranes to swell and loose their ribosomes. The replication of HPEV1 was found to take place on small clusters of vesicles which contained the trans-Golgi marker GalT as well as the viral non-structural 2C protein. 2C was additionally found on stretches of modified ER-membranes, seemingly not involved in RNA replication. The viral non-structural 2A and 2C proteins were studied in further detail and were found to display several interesting features. The 2A protein was found to be a RNA-binding protein that preferably binds to positive sense 3 UTR RNA. It was found to bind also duplex RNA containing 3 UTR(+)-3 UTR(-), but not other dsRNA molecules studied. Mutagenesis revealed that the N-terminal basic-rich region as well as the C-terminus, are important for RNA-binding. The 2C protein on the other hand, was found to have both ATP-diphosphohydrolase and AMP kinase activities. Neither dATP nor other NTP:s were suitable substrates. Furthermore, we found that as a result of theses activities the protein is autophosphorylated. The intracellular changes brought about by the individual HPEV1 non-structural proteins were studied through the expression of fusion proteins. None of the proteins expressed were able to induce membrane changes similar to those seen during HPEV1 infection. However, the 2C protein, which could be found on the surface of lipid droplets but also on diverse intracellular membranes, was partly relocated to viral replication complexes in transfected, superinfected cells. Although Golgi to ER traffic was arrested in HPEV1-infected cells, none of the individually expressed non-structural proteins had any visible effect on the anterograde membrane traffic. Our results suggest that the HPEV1 replication strategy is different from that of many other picornaviruses. Furthermore, this study shows how relatively small differences in genome sequence result in very different intracellular pathology.
  • Rajala, Hanna (Helsingin yliopisto, 2014)
    Large granular lymphocytic (LGL) leukemia is a chronic incurable disorder characterized by expansion of cytotoxic T- or natural killer (NK)-lymphocytes infiltrating blood and bone marrow. The diagnostic criteria include persistent lymphocytosis in peripheral blood, and in the case of T-LGL leukemia, detection of a clonal rearrangement of the T cell receptor. Typical clinical and hematological characteristics, including anemia, neutropenia, and autoimmune disorders, further support the diagnosis. Current immunosuppressive treatment options, such as methotrexate, induce remission in only 50% of cases and no targeted therapies exist. The aim of this project was to characterize the molecular pathogenesis of LGL leukemia and find molecular markers that could be used for diagnosis and novel therapeutic approaches. In the first study, the molecular background of LGL leukemia was analyzed by exome sequencing of a T-LGL leukemia patient. The index patient carried a novel D661V mutation in the Signal transducer and activator of transcription 3 (STAT3) gene. STAT3 is a known oncoprotein and a transcription factor. In the subsequent screening, the prevalence of somatic STAT3 mutations in T-LGL cases was 40%. Based on functional studies, the mutations located in the SRC-like homology 2 (SH2) domain increased phosphorylation and transcriptional activity of STAT3. A larger LGL-leukemia patient cohort, including both T-LGL leukemia and chronic lymphoproliferative disorder of NK cells (CLPD-NK) patients, was analyzed in the second study. Activating STAT3 mutations were seen in 27% and 30% of cases, respectively. The analysis of clinical characteristics showed that patients with STAT3 mutations were more likely to have neutropenia and rheumatoid arthritis and also required more frequent treatment when compared with unmutated patients. The third study concentrated on the analysis of STAT3 mutation-negative patients by exome sequencing. Two T-LGL leukemia patients carried Y665F mutation in STAT5B gene. In the screening of over 200 T-LGL leukemia and CLPD-NK cases, two patients presented with additional N642H mutation in STAT5B. The mutations were situated in the SH2 domain and led to increased phosphorylation and transcriptional activity of STAT5B. The in vitro effects of N642H mutation were more prominent, and both patients harboring N642H mutation had an untypically aggressive clinical presentation. In the fourth study, the clonal hierarchy and STAT3 mutation spectrum during immunosuppressive treatment was analyzed by deep amplicon-sequencing of STAT3 and simultaneous deep sequencing analysis of a T cell receptor beta chain (TCRB) repertoire. A total of 22% of STAT3-mutated patients harbored multiple STAT3 mutations mostly in separate lymphocyte clones. Mutated clones did not share common T cell receptor beta chain (TCRB) sequences by deep sequencing method in three T-LGL leukemia cases analyzed. Complete remission after immunosuppressive therapy resulted in the eradication of the STAT3-mutated clone in most patients, whereas partial responses reflected only modest changes in the leukemic clone burden. In conclusion, the activating STAT3 and STAT5B mutations provide a basis for understanding the molecular pathogenesis of leukemic LGL expansion. LGL leukemia can result from chronic antigen exposure in combination with immunogenetic factors such as STAT mutations. The detection of STAT3 and STAT5B mutations can be included in the diagnostic criteria of LGL leukemia, and facilitate development of novel therapeutics.
  • Aula, Nina (Helsingin yliopisto, 2003)