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Hexose-6-phosphate dehydrogenase: novel interactors and role in lipid metabolism
No full textHexose-6-phosphate dehydrogenase (H6PD) is the endoplasmic reticulum (ER) luminal counterpart of the cytosolic glucose-6-phosphate dehydrogenase (G6PD) and it catalyzes the first two steps of the pentose-phosphate pathway (PPP) in the ER, generating nicotinamide adenine dinucleotide phosphate (NADPH) in this process (Beutler & Morrison, 1967; Bublitz, 1981; Takahashi & Hori, 1978; Watanabe, 2017). To date, the only well-described interactor of H6PD is 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which utilizes luminal NADPH during the activation of glucocorticoids: cortisol and corticosterone in humans and rodents, respectively (Gathercole et al., 2013; Mziaut et al., 1999; Odermatt et al., 1999; Odermatt & Kratschmar, 2012). Increasing evidence suggests that H6PD plays a role in lipid metabolism. Lipolysis is impaired in adipose tissue and adipocyte-specific H6PD knock-out (KO) (Bujalska et al., 2008; Wang et al., 2019). It has also been demonstrated that upon fasting, no changes occur in the mobilization of fat and serum-free fatty acid (FA; pl. FAs) levels in H6PD KO mice, indicating the contribution of H6PD to lipolysis in adipose tissue (Bujalska et al., 2008). Therefore, H6PD appears to act as a bridge between the production of glucocorticoids and the metabolism of carbohydrates and lipids. However, despite increasing evidence of the function of H6PD in lipid metabolism of adipose tissue, the role of H6PD in the liver, which is a central hub for lipid metabolism, is poorly understood, and the exact mechanisms and pathways in which this protein participates remain to be discovered.
The first part of the thesis consists of a comprehensive review of H6PD, which discusses the role of this protein in health and disease. This review summarizes the molecular and biochemical properties of H6PD. Moreover, it provides a broad overview of the known functions of H6PD and its involvement in pathological processes. Particularly, the current knowledge regarding H6PD involvement in the luminal PPP and glucocorticoid activation is discussed. Moreover, H6PD role in skeletal muscle myopathy, metabolic disorders and cancer pathogenesis is reviewed in detail, presenting all available studies.
The second part of this thesis aims to elucidate novel interactors of H6PD in the ER lumen. To accomplish this aim, we took advantage of the BioID approach, which is a cutting-edge technique for identifying protein-protein interactions through proximity-based biotinylation. By employing BioID we identified 50 potential H6PD interactors. Among the identified H6PD interactome, protein disulfide isomerase (PDI) family member anterior gradient protein 2 (AGR2) was found. AGR2, as well as H6PD, has been previously indicated to contribute to breast cancer progression. Therefore, we validated the interaction by co-immunoprecipitation. Additionally, gain of function and loss of function studies provided evidence that AGR2 regulates H6PD activity. Finally, mRNA expression analysis of AGR2 and H6PD using the Cancer Genome Atlas (TCGA) database was performed. Analysis of H6PD expression in breast cancer tumors compared to the normal tissue indicated that H6PD expression is lower in malignant tissue. Further analysis provided evidence that H6PD expression positively correlates with the survival of triple-negative patients. Additionally, AGR2 expression in estrogen receptor-expressing tumors correlated with better survival. Overall, this study confirmed the applicability and reliability of the BioID approach in the ER compartment and helped to identify novel H6PD interactors.
In the final part of this thesis, we aimed to understand the role of H6PD in hepatic lipid metabolism. Increasing evidence suggests H6PD function in lipid metabolism of adipose tissue. However, the role of H6PD in the liver, which is a central hub for lipid metabolism, is poorly understood, and the exact mechanisms and pathways in which this protein participates remain to be discovered. Therefore, we aimed to understand the function of H6PD in lipid metabolism pathways in the liver. First, we employed lipidomics approach to compare lipid profiles of wild type (WT) and H6PD KO mouse liver tissue. Lipidomics analysis indicated increased accumulation of long chain, low saturation triacylglycerol (TG) when H6PD is lacking. Next, we validated increased TG accumulation in H6PD KO mouse liver tissue sections and AML12 mouse hepatocytes. To reveal lipid metabolism pathways that are affected by H6PD KO, we employed proteomics-based pathway analysis of WT and H6PD KO mice liver tissue. Pathway analysis revealed that H6PD KO influences multiple aspects of lipid metabolism. Subsequently, based on multi-omics findings, we formulated a hypothesis and experimentally investigated changes that could lead to increased accumulation of TG in H6PD KO liver tissue and cells. We observed that H6PD KO AML12 cells shift from glucose to FA dependency, accompanied by increased extracellular uptake of FAs and increased palmitic acid PA-induced cytotoxicity. Our analysis revealed a downregulation of key enzymes involved in mitochondrial FA β-oxidation (mtFAO), including carnitine palmitoyltransferase 1 A and B (CPT1A; CPT1B), and carnitine palmitoyltransferase 2 (CPT2) in both H6PD KO liver and AML12 cells. A notable decrease in mtFAO in H6PD KO AML12 cells was observed. Furthermore, we demonstrated that CPT1 downregulation and the subsequent decrease in mtFAO occurs through the adenosine monophosphate (AMP)-activated protein kinase alpha subunit (AMPKα) and acetyl-CoA carboxylase 1 (ACC1) axis. Therefore, apart from playing a critical role in glucocorticoid production, H6PD is also involved in lipid homeostasis, regulating FA import and metabolic processes.
In summary, the studies described in this thesis provided valuable insights regarding the H6PD interactome and the involvement of this protein in lipid metabolism pathways. The combination of complex techniques, such as BioID, lipidomics, and proteomics analysis combined with functional assays, were useful in achieving the goals of this thesis. However, many aspects regarding H6PD interactions and functional roles still remain to be answered
Molecular mechanisms of mRNA stability: the impact of codon optimality and temperature
No full textIn the grand scheme of gene regulation, RNA sits at the centre, being the metaphorical ‘middle man’ in the transfer of information from DNA to protein. This makes mRNA an important target for gene regulation processes. Gene expression can be controlled at the mRNA level by regulating transcription (affecting production), causing sequestration of mRNA (affecting local concentration), regulating mRNA decay rates, or impacting production of protein by translation. In this thesis, I study mRNA from three angles: codon dependence of mRNA stability, temperature dependence of mRNA stability and temperature dependence of hyphal transition in Candida albicans.
Firstly, I examine the molecular underpinnings of the linkage between codon optimality and mRNA half-lives. A link between mRNA decay and translation has been a common theme in several studies with optimal codons being reported to increase translation rates and lead to longer half-lives. In our investigation, we found that there is a threshold length, only above it do cells respond to codon optimality with differential mRNA stability. This threshold length corresponds to the preferential translation of the transcripts by polyribosomes and the untranslated regions of the mRNA were found to determine the position of this threshold length, or whether it will exist at all.
Secondly, I study the degradation and translation dynamics of mRNAs under heat shock as compared to standard temperatures. Accurate measurements of mRNA half-lives have been few and far between: more so at elevated temperatures. In addition, the datasets resulting from these studies have shown strong method-to-method variations and in several cases, even different studies using the same method have led to uncorrelated half-lives. I used approach to equilibrium technique of RNA metabolic labelling to generate high quality RNA half-life datasets in S. cerevisiae at standard and elevated temperatures. They show high inter-temperature correlation as well as high correlation with external measurements. In addition, I examine the translation status of transcripts at standard and heat shock temperatures in yeast and mammalian systems.
The third part of this thesis chronicles our search for a temperature dependent switch in RNA turnover in Candida, which would lead to hyphal induction. The hyphal transition is important for pathogeny and thus clinically relevant. I show that temperature alone is sufficient for the hyphal transition and that several RNA markers can predict this transition, before the morphology appears
Das Verbot der Embryonenspende in der Schweiz: eine rechtliche Analyse des Verbots der Spende «überzähliger Embryonen» zu Fortpflanzungszwecken
Get PDFIm Rahmen der In-vitro-Fertilisation (IVF) werden üblicherweise mehrere Embryonen erzeugt, von denen ein bis zwei zur Herbeiführung einer Schwangerschaft in die Gebärmutter transferiert werden. Nicht sofort transferierte Embryonen können für spätere Versuche konserviert werden. Nach abgeschlossener Kinderwunschbehandlung sind möglicherweise noch konservierte Embryonen vorhanden, welche das Kinderwunschpaar nicht mehr verwenden kann oder will. In der Schweiz müssen diese „überzähligen Embryonen“ vernichtet werden. Die Embryonenspende, also die Spende von überzähligen Embryonen zu Fortpflanzungszwecken an andere Kinderwunschpaare, ist nach Art. 119 Abs. 2 lit. d BV und Art. 4 FMedG unzulässig. Das vorliegende Werk untersucht, weshalb die Embryonenspende verboten ist und ob insbesondere im Lichte der neuen Entstehungsmöglichkeiten überzähliger Embryonen («Zwölferregel») dieses Verbot reformiert oder sogar gänzlich aufgehoben werden muss
Spinal cord gray and white matter atrophy in patients with motor neuron diseases
No full textMotor neuron disorders (MND) are a heterogeneous group of disease of the upper (UMN) and lower motor neurons (LMN) with a wide spectrum of etiologies.
Among them, Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative MND leading to the decline of the UMN and LMN and thus, to progressive muscular spasticity and weakness. Death usually occurs from respiratory failure. Despite ever-increasing efforts in last years, there are no curative therapeutic options and disease-prolonging therapies are sparse.
In order to identify potentially effective new treatment options, valid and reliable biomarkers are needed to assess disease progression and to evaluate the effect of new therapeutic approaches. Imaging markers may serve this purpose, but so far, reliable and easily implementable imaging biomarkers for ALS are lacking. The radially acquired Averaged Magnetization Inversion Recovery Acquisitions (rAMIRA) is a novel Magnetic Resonance Imaging (MRI) technique that enables high-resolution imaging of the spinal cord (SC) gray (GM) and white matter (WM) in clinically feasible acquisitions times. Using
rAMIRA imaging, GM and WM metrics may have the potential to become new imaging markers in patients with MND.
This PhD thesis comprises three studies evaluating SC metrics in patients with MND, evaluating their potential as new imaging markers. As a first step, we investigate the SC GM atrophy as a surrogate for LMN damage in a presumably pure LMN disorder, the Post-Polio Syndrome (PPS). This work was the first implementation of the rAMIRA sequence in an MND. By essentially using PPS as a model disease, this first work built a basis for the implementation of rAMIRA in other MND. As a second step, both SC GM and WM were evaluated in a combined UMN and LMN disorder, i.e. ALS. As a third step, a new MRI sign for ALS was evaluated in patients with ALS and other non-ALS MND.
In the first study, we evaluated the SC GM in patients with PPS. We compared the cervical and thoracic SC GM of patients with PPS to healthy, age- and sex-matched controls (HC) and assessed the relationship between the SC GM at different intervertebral disc levels and the segmental muscle strength at the respective corresponding myotomes. We were able to demonstrate significant cervical and thoracic SC GM atrophy in patients with PPS and found significant correlations between SC GM areas and muscle strength at corresponding myotomes. This study provided an encouraging first step for the implementation of the rAMIRA sequence in further MND.
In the second study, we evaluated both the SC GM and WM in patients with ALS, which is a combined disorder of the UMN and LMN. Similar to the first study, we compared the cervical and thoracic SC GM and WM of patients with ALS to age- and sex-matched HC and assessed the relationship of SC GM and WM at cervical and thoracic levels with segmental muscle strength. Moreover, we analyzed the relationship between the SC GM and WM and established markers of disease progression. We were able to demonstrate significant atrophy of cervical and thoracic SC GM and WM compared to controls, and found significant correlations between GM atrophy and muscle strength at corresponding myotomes as well as significant correlations with markers of respiratory strength and overall disability.
The third study describes and evaluates an observation we made during the analyses of the data from the second study: We observed a hyperintensity of the SC corticospinal lateral tracts on rAMIRA imaging in a patient with ALS who clinically presented a predominant UMN disease type. We then observed that these hyperintensities were visible in patients with, but also in some patients without a predominant UMN ALS. Building on these observations, we identified, defined, and analyzed the presence of the sign in a group of patients with ALS, a group of HC and a control group comprised of patients with non-ALS LMN disorders. The sign showed a good sensitivity and an excellent specificity for the diagnosis of ALS. Furthermore, we performed post-mortem imaging and consecutive histopathological analysis of one patient with ALS and an UMN predominant disease type. The histopathological analysis showed a rarefaction of myelinated axons in the region of the hyperintensities observed on rAMIRA imaging.
Taken together, the results from these three projects suggest that SC metrics acquired using rAMIRA imaging show promise as potential future imaging markers in patients with MND
Niche differentiation between some species of Ghanaian orb-web spiders (Araneae: Araneidae, Tetragnathidae)
No full textThe web placement and web structure of the nine most common species of orb-web spiders (Araneae: Araneidae, Tetragnathidae) coexisting in the park-like campus of the University of Ghana in Legon, Ghana, were investigated to analyse differences between their ecological niches. Various web parameters, such as: relation to habitat, height, light exposure, web size, mesh size, and web inclination, were measured. When comparing these parameters in all species pairs, the webs of each species were found to differ in at least two parameters from the web of every other species. Since the analysed web parameters are all likely to influence the number and kind of prey caught, this suggests a niche differentiation between all analysed species. Some observations were also made on the prey caught. While the prey analysis was not detailed enough to find differences between all species, there was a positive correlation between the web's mesh size and the size of the prey
Structural and functional analysis of the human chemokine receptor and HIV-1 co-receptor CCR5
Get PDFThis thesis describes the elucidation of the activation mechanism of the human CC chemokine receptor 5 (CCR5) by a chemokine analog, its signaling complexes with the heterotrimeric Gi protein, and subsequent interactions with arrestin2.
Chapter 1 presents a general introduction to G protein-coupled receptor (GPCR) biology and recent advances in the structure elucidation of GPCRs and their signaling complexes. Additionally, the chapter covers the biology of chemokine receptors, focusing on the role of CCR5 in the human immune system.
Chapter 2 describes the cryo-EM structure of wild-type human CCR5 in an active conformation bound to the super-agonist [6P4]CCL5 and the heterotrimeric Gi. The structure of the signaling complex allowed to describe the activation mechanism of CCR5 and to elucidate key elements of the variable pharmacology of CCL5 analogs. These results shed new light on the molecular pharmacology of chemokine receptors and show how a chemokine receptor can be activated by the ‘deep’ binding of the agonist N-terminus into the orthosteric receptor pocket.
Chapter 3 provides a detailed protocol for the biochemical preparation of the [6P4]CCL5•CCR5•Gi signaling complex and describes current advances in chemokine structure determination and associated challenges. An additional NMR characterization of the [5P14]CCL5 partial agonist and [5P12]CCL5 antagonist chemokines is given in the Appendix of this Chapter.
Chapter 4 focuses on the last step of the GPCR signaling cascade – the interaction of CCR5 with arrestin2. Two high-resolution X-ray structures of human arrestin2 in complex with two distinct CCR5 C-terminal phosphopeptides were solved. These structures, in combination with mass spectrometry, NMR, and biochemical and cellular assays, uncovered a key GPCR phosphomotif, which is recognized by arrestin2 and enables its tight association with a GPCR. A further analysis of available structural and functional data on GPCR•arrestin interactions suggests how a certain arrangement of phosphoresidues within the intracellular side of GPCRs define arrestin2 and arrestin3 isoforms specificities.
Chapter 5 describes the isolation of the full-length [6P4]CCL5•CCR5•arrestin2 complex, its biochemical and structural characterization. Furthermore, it discusses the challenges of the structural analysis of this complex
Artificial intelligence and the doctor-patient relationship expanding the paradigm of shared decision making
Get PDFArtificial intelligence (AI) based clinical decision support systems (CDSS) arebecoming ever more widespread in healthcare and could play an important role indiagnostic and treatment processes. For this reason, AI‐based CDSS has an impacton the doctor-patient relationship, shaping their decisions with its suggestions. Wemay be on the verge of a paradigm shift, where the doctor-patient relationship is nolonger a dual relationship, but a triad. This paper analyses the role of AI‐based CDSSfor shared decision‐making to better comprehend its promises and associated ethicalissues. Moreover, it investigates how certain AI implementations may instead fosterthe inappropriate paradigm of paternalism. Understanding how AI relates to doctorsand influences doctor-patient communication is essential to promote more ethicalmedical practice. Both doctors' and patients' autonomy need to be considered in thelight of AI
Modelling gene expression in terms of DNA sequence
Get PDFUnderstanding the gene regulatory networks that control gene expression remains one of the most of important questions in molecular biology. Much of gene expression is controlled through transcription initiation, whose regulation is ultimately encoded in the constellations of small sequence motifs in the DNA that are bound by transcription factors (TFs) in a sequence-specific manner. In this thesis, we addressed the task of understanding gene regulation on two levels. Firstly, we present a computational pipeline for inferring a set of gene regulatory elements in a given organism which includes identifying genes that encode DNA-binding domains (DBDs), mapping them to known binding motifs by leveraging similarity in DBDs between species, annotating promoter regions genome-wide, aligning promoters with orthologous regions from related genomes, and predicting genome-wide transcription factor binding sites (TFBSs). We demonstrated the use of our pipeline by applying it to zebrafish. Furthermore, we integrated these results into our previously developed Integrated System for Motif Activity Response Analysis (ISMARA) which models gene expression data in terms of predicted regulatory sites. Using ISMARA, we predicted known and novel key regulatory TFs in zebrafish using a number of RNA-seq datasets. Secondly, we zoom in at the scale of one single TF regulating a set of constitutive promoters in \textit{Escherichia coli}. We analyzed an artificially evolved set of synthetic promoter sequences which are selected for expression constitutive promoters regulated by transcription factor. We looked closely into promoter sequences and TF binding dynamics and investigated the predictive power of TF binding affinity on gene expression
