Perturbed rhythmic activation of signaling pathways in mice deficient for Sterol Carrier Protein 2-dependent diurnal lipid transport and metabolism

Through evolution, most of the living species have acquired a time keeping system to anticipate daily changes caused by the rotation of the Earth. In all of the systems this pacemaker is based on a molecular transcriptional/translational negative feedback loop able to generate rhythmic gene expression with a period close to 24 hours. Recent evidences suggest that post-transcriptional regulations activated mostly by systemic cues play a fundamental role in the process, fine tuning the time keeping system and linking it to animal physiology. Among these signals, we consider the role of lipid transport and metabolism regulated by SCP2. Mice harboring a deletion of the Scp2 locus present a modulated diurnal accumulation of lipids in the liver and a perturbed activation of several signaling pathways including PPAR alpha, SREBP, LRH-1, TORC1 and its upstream regulators. This defect in signaling pathways activation feedbacks upon the clock by lengthening the circadian period of animals through post-translational regulation of core clock regulators, showing that rhythmic lipid transport is a major player in the establishment of rhythmic mRNA and protein expression landscape.

The bile acid membrane receptor TGR5: a valuable metabolic target

Johan Auwerx, Mitsunori Nomura, Lilia Noriega Lopez, Thijs Pols, Kristina Schoonjans

Bile acids (BAs) are amphipathic molecules that facilitate the uptake of lipids, and their levels fluctuate in the intestines as well as in the circulation depending on food intake. Besides their role in dietary lipid absorption, BAs function as signaling molecules that activate specific BA receptors and trigger downstream signaling cascades. The BA receptors and the signaling pathways they control are not only important in the regulation of BA synthesis and their metabolism, but they also regulate glucose homeostasis, lipid metabolism and energy expenditure - processes relevant in the context of the metabolic syndrome. In addition to the function of the nuclear receptor FXRα in regulating local effects of BAs in the organs of the enterohepatic axis, increasing evidence points to a crucial role of the G-protein-coupled receptor TGR5 in mediating systemic actions of BAs. Here we review the current knowledge on BA receptors, with a strong focus on the cell membrane receptor TGR5, which has emerged as a promising target for intervention in metabolic diseases.

2011

In Vivo Studies on the Control of Gene Expression and Protein Degradation

Karolina Bojkowska

Cellular homeostasis is maintained by tightly regulated gene networks, controlled notably at the levels of transcription, mRNA processing, and protein post-translational modification and turnover. This thesis focuses on two of these regulatory steps, namely gene transcription and protein stability. I first explored the impact of the KRAB-ZFP/KAP1 gene regulation system on liver function. KRAB-ZFPs constitute the largest family of transcription factors encoded by mouse and humans, yet their physiological roles and gene targets remain mostly elusive. Conversely, their molecular mechanism of action has been rather well characterized, as they can recognize DNA in a sequence-specific manner and recruit the universal co-factor KAP1 (also known as TRIM28), which in turn serves as a scaffold for various heterochromatin-inducing effectors. Several studies have shed some light on the roles played by this system in embryonic tissues, but its functions in the adult remain almost completely unknown. As chromatin regulation has been shown to be a major factor in the control of metabolism, we decided to investigate the role of KRAB-ZFP/KAP1 in the liver, an organ central to this process. For this, we generated conditional liver-specific Kap1 KO mice, the study of which revealed a strikingly sex-specific phenotype, with early-onset steatosis and age-related tumorigenesis restricted to males, contrasting with the mild metabolic disturbances recorded in Kap1-deleted females. Underlying this phenotype, our combined transcriptome and chromatin analyses revealed that KAP1 controls sexually dimorphic genes notably involved in hormone, drug and xenobiotic metabolism. Finally, by using a recently developed technique for direct RNA quantification, we established the range of KRAB-ZFPs expressed in the liver and likely responsible for at least part of the observed effects. This study thus demonstrates the central role of the KRAB/KAP1 system in control of sexual dimorphism, responses to xenobiotic stress and metabolic control in the liver, and further links disturbances in these processes with hepatic carcinogenesis. In parallel, in an effort to pursue a multidisciplinary training combining skills in biology and chemistry, I worked towards the development of a method to study the half-life of proteins in vivo. Protein turnover critically influences many biological functions, yet methods have been lacking to assess this parameter in vivo. Capitalizing on an in vitro technology previously developed in Kai Johnsson's laboratory, I demonstrated how chemical labeling can be used to measure the half-life of resident intracellular and extracellular proteins in living mice. Our approach is based on labeling of SNAP-tag-fusion proteins with fluorescent probes. First, we verified that SNAP-tag substrates have wide bio-availability in mice and can be used for the specific in vivo labeling of SNAP-tag fusion proteins. We then applied near-infrared probes to perform non-invasive imaging of in vivo labeled tumors. Finally, we used SNAP-mediated chemical pulse-chase labeling to measuring in vivo the half-life of different extra- and intracellular proteins, including KAP1. Importantly, this tagging technology can be applied to any protein amenable to expression as a fusion partner, whether cell-associated or secreted. Furthermore, because the SNAP-tag chemical labeling allows for a large array of probes also suited for experiments aimed at manipulating and monitoring protein function in vivo, such as cross-linking, pull-down or FRET, this methodology opens broad perspectives for studying protein function in living animals.

EPFL2011

Towards a kinetic model of the phospholipids biosynthesis of the yeast Saccharomyces cerevisiae

Meriç Ataman, Vassily Hatzimanikatis, Iro Oikonomidi, Georgios Savoglidis

Lipids, as vital eukaryotic cellular components, play a major role in cell, tissue and organ physiology. Alterations of lipid enzymes and metabolic pathways are related to many human diseases, such as cancer, diabetes and neurodegenerative diseases. In this study, we focus on phospholipids, the major components of all cell membranes, which not only act as structural membrane constituents but also play a vital role in cellular energy storage and in signaling pathways. The yeast, Saccharomyces cerevisiae, has been chosen as a cell model, due to its strong homology of proteins and pathways with higher eukaryotes. The benefits of studying the glycerophospholipid metabolism of S. cerevisiae will provide critical insight into phospholipid homeostasis of this model organism in various cellular states. The reconstructed model of phospholipid metabolism consists of 118 reactions and 80 metabolites. Beyond combining knowledge of all published yeast genome-scale models, it incorporates missing reactions reported in the literature along with the current state of the art concerning the participating enzymes. Pathways regarding compounds from the glycolysis pathway and synthesis of acyl-CoAs, required for phospholipid biosynthesis, have also been included. A steady state flux analysis was performed with the growth capacity of the cell being able to vary between maintenance and a maximum experimentally observed growth rate, in aerobic carbon-limiting conditions, with mass balance constraints calculated based on experimental data reported in the most recent yeast genome scale model. A thermodynamic curation of all the reactions of the pathway followed, which constrains the allowed space of the concentration ranges of metabolites of the pathway and also provides information about the directionality of all the reactions. Finally, we applied the ORACLE methodology in order to identify enzymes that determine the lipid composition in the various steady states of the cell. Generating a comprehensive kinetic model will be a useful tool for predictive analysis, drug designing or further biomedical research.