糖心Volg

Roger Schneiter

photo

 roger.schneiter@unifr.ch
 +41 26 300 8654
 

Professeur路e ordinaire
D茅partement de biologie

PER 07 bu. 0.307
Ch. du Mus茅e 6
1700 Fribourg
PER 07, 0.307
+41 26 300 8654

Recherche et publications

  • Publications SchneiterLab
    117 publications


    Forests (2025) | Article

  • Projets de recherche

    10000909 - Unveiling the Function and Mechanisms of Pathogenesis-Related (PR)-Like Proteins Produced by Phytopathogens
    Statut: En cours

    顿茅产耻迟 01.05.2024
    Fin 31.08.2027
    Financement FNS 
    Plants are vital members of our ecosystem and form the basis of our food-chain. Their health and proliferation are threatened by phytopathogens and abiotic stress factors, including drought and climate change. In response to these challenges, plants have evolved a sophisticated multilayered defense system capable of detecting pathogens and restricting their propagation. At the heart of this defense system lies the induction of pathogenesis-related proteins (PRs), a group of defense-related proteins. These proteins collectively establish an antimicrobial network that aids in countering threats. PR proteins consist of 17 distinct families (PR1-PR17), including enzymes like 脽-1,3-glucanase (PR2) and various chitinases (PR3, PR4, PR8, PR11), alongside lesser-characterized proteins such as the CAP family protein (PR1), and a putative aminopeptidase (PR17). These proteins are typically secreted into the apoplast, an essential battleground in plant-microbe interactions. Overexpressing these proteins enhances pathogen resistance, while their downregulation weakens plant immunity. Recent findings have unveiled that not only plants synthesize PR proteins, but pathogens like helminths, fungi, and oomycetes also secrete similar PR-like proteins into the apoplast upon infecting their host plants. Intriguingly, downregulating PR-like proteins in pathogens reduces virulence, while their overexpression in plants heightens susceptibility to pathogen attacks. This indicates that these pathogen-derived PR-like proteins bolster pathogen virulence while compromising the plant's immune response. The fundamental question that we aim to address in this line of research is: Is the observation that PR-like proteins from pathogens enhance virulence and attenuate host immunity a general trait shared by all such proteins? If so, what explains the divergent effects of proteins from a same family, depending on whether they originate from the host plant or the invading pathogen? To tackle these queries, the genomes of important model fungal and oomycete pathogens (Botrytis cinerea, Fusarium oxysporum, Phytophthora infestans) were analyzed for PR-like genes. Results indicate a prevalence of PR-like gene homologs in pathogens. This study focuses on a subset of PR-like proteins including PR1-like (CAP family), PR5-like (thaumatin), and PR7-like (subtilisin-like endoprotease) proteins. The aim is to assess the impact of their downregulation in different pathogens on virulence, and conversely, to assess whether their heterologous expression in plants attenuates the host's immunity. Our working hypothesis is that PR-like proteins could act as decoys to disrupt the function of native PR proteins. To test this, investigations will focus on identifying PR-like protein interactions with host proteins in the infected tissue, as well as characterizing these interactions using purified components in vitro. The outcomes of this study promise to enhance our comprehension of host-pathogen interactions, particularly the intriguing juxtaposition of plant-derived PR proteins and pathogen-produced PR-like proteins. Given the growing interest in transgenic plants overexpressing PR proteins for reinforcing resistance against biotic and abiotic stressors, these findings could prove important in advancing crop protection strategies.

    Biogenesis and Turnover of Lipid Droplets
    Statut: En cours

    顿茅产耻迟 01.12.2022
    Fin 30.11.2026
    Financement FNS 
    Most cells store an excess of metabolic energy in form of neutral lipids within a dedicated intracellular compartment, known as lipid droplet (LD). This organelle is composed of a hydrophobic core of neutral lipids, mainly triacylglycerol (TAG) and steryl esters (STE), which is shielded from the aqueous environment by an unusual phospholipid monolayer, and functionalized by a specific set of LD-localized proteins. Many LD proteins directly act in neutral lipid synthesis and/or turnover, for example as acyl transferases or lipases, others, such as the perilipin class of proteins that stabilize the LD surface and also function as regulators of lipolysis. LDs not only serve to store energy, but they also provide an essential buffering capacity of lipid precursors to allow for rapid membrane expansion during cell division or expansion of intracellular organelles, for example during starvation and onset of autophagy. LDs are thus intimately linked to basic cellular processes including stress response, and their homeostasis is affected in prevalent human diseases including obesity, atherosclerosis, and diabetes. LDs are closely associated with the membrane of the endoplasmic reticulum (ER) with which they exchange proteins and lipids. They are formed at highly specialized ER subdomains where the LD biogenesis factor seipin colocalizes with a phosphatase complex that regulates the production of diacylglycerol (DAG), the rate-limiting precursor of TAG formation. Seipin forms a large membrane embedded oligomeric toroidal structure. Colocalization of seipin with the phosphatase complex not only ensures the local production of DAG but also results in recruitment of the TAG biosynthetic enzymes as well as accessory proteins, thereby allowing for localized channeling of TAG into growing LDs. Perturbation of this spatially and temporally highly defined assembly of LD biogenesis proteins results in the ectopic formation of aberrant, non-functional LDs throughout the ER membrane and the induction of an ER stress response. The aim of the present research project is to characterize this spatial and temporal regulation of LD formation and turnover in the ER and to identify and characterize both cis- and trans-acting factors, including regulatory components, that are required for these processes. What are the molecular cues that result in the colocalization of seipin with the phosphatase complex and how exactly is the local formation of DAG and TAG regulated? These questions will be addressed by using time-resolved biochemical and microscopic readouts in wild-type cells as well as in mutant cells lacking individual components of the LD assembly complex of proteins. In addition, we will perform a detailed structural and functional analysis of one of the trans-acting LD assembly factors, the perilipin (PLIN) family member PLIN3. We have evidence that this soluble protein induces the formation of membrane domains enriched in both DAG and seipin, and we will use liposome-binding assays and cryo-electron microscopy to characterize the lipid-binding and possible membrane-coat forming properties of PLIN3. In an independent line of investigations, we will characterize the activation and regulation of LD-localized lipases to understand how the action of these lipases and the degradation of LDs is controlled. We have identified kinase and phosphatase mutants in which the turnover of TAG, that of STE, or that of both neutral lipid classes is blocked and we will analyze how these mutants impinge on the activation of the TAG and STE lipases. These studies will help understand the molecular processes that underlie and coordinate the biogenesis and turnover of LDs and will reveal important regulatory factors in these two antagonistic processes, lipogenesis and lipolysis.

    Deciphering the functions of the conserved fat storage-inducing transmembrane (FITM2) protein in lipodystrophy and insulin resistance through systems approaches
    Statut: En cours

    顿茅产耻迟 01.09.2021
    Fin 31.08.2025
    Financement FNS 
    The metabolic syndrome has reached a pandemic scale affecting about 20-25% of the world鈥檚 adult population. This syndrome is characterized by a strong elevation of the body mass index, mostly due to accumulation of adipose tissue, the main tissue dedicated to the storage of fat. While the unprecedented increase of this condition is due in part to malnutrition and lack of physical activity, there are also genetic predispositions that affect fat metabolism and storage in organs, cells, and even at a molecular level. Within adipocytes, fat is stored in a dedicated globular compartment termed lipid droplet (LD). LDs are present in most cells of the body, including skeletal and cardiac muscle cells. However, in adipocytes they are particularly large, unilocular, and they fill most of the cell volume. LDs constitute a functionally conserved fat storage compartment found not only in metazoans but also in bacteria, fungi and plant cells. Structurally, LDs are unique in that they are composed of a globular core of neutral lipids (i.e., fat), which is enclosed by a phospholipid monolayer to shield the hydrophobic interior from the aqueous environment. LDs are thus structurally related to lipoprotein particles, but unlike these, they are not secreted. The compartment is functionalized by proteins and enzymes that specifically localize to the LD surface. Many of these LD-localized proteins directly operate in neutral lipid synthesis or degradation, for example as acyltransferases or lipases. The molecular mechanisms that regulate fat storage during lipogenesis and lipolysis, however, are not well understood. Defects in LD formation result in severe pathologies including lipodystrophy, which is characterized by an abnormal distribution of body fat, ectopic fat accumulation (HIV-related), and insulin resistance. Insulin resistance can thus be caused by both a lack as well as a surplus of body fat, and hence is closely related to neutral lipid metabolism, endoplasmic reticulum (ER) stress, and inflammation. LDs are formed from the ER membrane with which they maintain a close association and exchange both proteins and lipids. The formation of LDs is driven by ER-localized enzymes that catalyze the synthesis of neutral lipids, particularly triacylglycerol and cholesteryl ester. Initially, these neutral lipids form small lens-like structures in the ER membrane, which grow in size and mature to nascent LDs. While remaining connected to the ER membrane, nascent LDs further mature in their protein composition and grow until they emerge towards the cytoplasm. The aim of the present proposal is to characterize early steps in LD biogenesis, i.e., the partitioning of neutral lipids between the ER membrane and LDs. This partitioning requires a conserved class of ER resident integral membrane proteins known as fat storage-inducing transmembrane (FIT2/FITM2) protein. Generally, a decrease of FITM2 function results in lower levels of neutral lipids and hence a reduction in number and size of LDs, whereas overexpression of these proteins results in the reverse, i.e., neutral lipid accumulation and an increased number and size of LDs. In higher eukaryotes FITM2 function is essential and its postnatal deletion in mice results in intestinal fat accumulation whereas its overexpression affects skeletal muscle energy homeostasis. In yeast cells, lack of FITM2 results in aberrant LD formation as LDs emerge towards the ER lumen rather than the cytoplasm. Here, we will employ FITM2 as an entry point to gain a deeper insight into the early stages of LD biogenesis, particularly the sequestration of TAG from the ER bilayer into the hydrophobic core of LDs, and to identify key proteins and metabolic pathways that impinge upon neutral lipid storage within LDs. We will take systems-wide approaches to understand the function of FITM2 proteins in fat metabolism. For this purpose, we will generate both mammalian and yeast cell lines in which FITM2 is depleted. The immediate early consequences of this lack of FITM2 function will then be analyzed at different omics-levels by characterizing acute changes in gene expression (through RNA-Seq analysis), protein abundance (through stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics), changes in the phosphorylation pattern of proteins (through SILAC-based phosphoproteomics analysis), and changes in the lipidome (following stable isotope labelling). In addition, we will screen for genes that genetically interact with FITM2 in mammalian cells through a CRISPR interference screen (CRISPRi, clustered regulatory interspaced palindromic repeats) and in yeast cells using a saturated transposon array (SATAY) approach. These systems-level approaches will be complemented by more directed hypothesis-driven approaches through which we will identify proteins that directly interact with FITM2 (through co-immunoprecipitation and proximity labelling) and characterize the function of FITM2 during lipogenesis. In addition, we will investigate early steps of LD formation in cells lacking FITM2 by both electron- and fluorescence-microscopy to analyze whether other known factors in this process such as Seipin, the causative agent of Berardinelli-Seip disease, Lipin, the rate-limiting enzyme in triacylglycerol formation, or the membrane shaping protein Pex30/MCTP2 are aberrantly localized. We have previously shown that LD formation is a highly localized and ordered process, restricted to subdomains of the ER membrane. Absence of any of the LD biogenesis factors from these ER subdomains results in random triacylglycerol synthesis and ectopic droplet formation. Ectopically formed LDs, however, are functionally impaired as they lack the full complement of LD surface proteins, such as lipases required for neutral lipid turnover, and thus may induce ER stress and render cells susceptible to lipotoxicity. Integrating the resulting systems-wide datasets with the results of the hypothesis-driven approaches will facilitate the identification of cellular pathways that depend on FITM2 function and thus yield insights into the etiology of neutral lipid storage disorders. Candidate pathways and components identified through these approaches will be tested further for their function in neutral lipid metabolism and LD formation. This is a collaborative project between principle investigators (PI) located in Switzerland and India. The two PIs know each other since more than 15 years and have published important papers in the field of LD biogenesis together. They both have a track record in lipid metabolism and LD biogenesis and a complementary technical background. The group in India will focus on FITM2 function in mammalian cells and perform all electron microscopy experiments needed for this collaboration. The group in Switzerland, on the other hand, will perform the genetic screen in yeast cells and perform the mass-spectrometry-based analyses. Both groups will perform immunoprecipitation experiments, analyze the function of FITM2 in lipogenesis in their respective model systems, and conduct the fluorescence-based colocalization studies. This is a comprehensive systems-wide approach to gain a better understanding of a medically important process, the storage and utilization of fat at the cellular level. Understanding the cellular regulation of the process is a prerequisite for an improved understanding at the organismal level. Moreover, the causal relations of molecular processes are more amenable at the cellular level than at the organismal level, where systems-level complexity frequently becomes overwhelmingly convoluted and hence causal relations can be masked. Funding of this project would allow the two PIs to establish a fruitful collaboration on a timely and medically important topic and break new grounds in LD biogenesis.

    Deciphering pathways of fatty acid efflux and their use to optimize microbial cell factories
    Statut: Termin茅

    顿茅产耻迟 01.02.2019
    Fin 31.01.2024
    Financement FNS 
    The question addressed in this research proposal is: how are fatty acid exported from cells? This basic question is of high medical and applied importance. It is of medical importance because the fat storing adipocytes of humans release large quantities of free fatty acids into the circulation upon starvation induced lipolysis, i.e., the lipase-mediated degradation of fat depots. This lipolytic pathway and its resulting accumulation of free fatty acids is intimately linked to metabolic disorders such as obesity, type 2 diabetes and chronic inflammation. The question is of applied relevance because there is a large commercial interest in establishing sustainable sources of biofuel production, i.e., the production of fatty acid derivatives through microbial fermentation. Given the importance of this fatty acid efflux pathway, it is surprising to realize how little we actually know about it. We essentially only know that fatty acid efflux occurs but, we do not know how it occurs at a genetic and molecular level: no transporters or carriers have yet been identified. The aims of the project are twofold. First, we seek to identify molecular components that are required for fatty acid efflux to occur. We hope to achieve this by performing a genetic screen in baker鈥檚 yeast, Saccharomyces cerevisiae, to isolate mutants that are synthetic lethal or synthetic sick under conditions when export of free fatty acids is blocked, leading to their toxic accumulation. This genetic screen will be complemented by a more analytical approach to identify genes and their products being upregulated in the oleaginous yeast species, Starmerella bombicola and Yarrowia lipolytica under conditions where these yeasts secrete large quantities of fatty acids into the culture medium. Candidate genes will then be downregulated and the time-dependent intracellular accumulation of free fatty acids will be monitored. This ab initio approach will be complemented by a candidate gene approach in which we will test whether small secreted fatty acid-binding proteins play a role in the efflux pathway. Our preliminary data indicate that depletion of a small secreted fatty acid-binding protein indeed halts the efflux of free fatty acids. The second aim of the project is to apply the knowledge gained from the above-mentioned approaches to improve the efficiency of fatty acid secretion and thus the overall yield of fatty acids that can be produced by S. cerevisiae and oleaginous yeasts. If this can be achieved, we will aim at modifying the substrate specificity of the identified efflux components to allow for tailored recognition of related small hydrophobic components whose biotechnological production is of relevance, such as fatty alcohols, dicarboxylic acids and polyunsaturated fatty acids (PUFAs).

    Lipid Storage, Binding, and Export
    Statut: Termin茅

    顿茅产耻迟 01.12.2017
    Fin 30.11.2022
    Financement FNS 
    This research program addresses two separate topics: the biogenesis of lipid droplets and their association with the membrane of the endoplasmic reticulum, and the structure and function of an important and widespread protein superfamily, known as CAP proteins. Lipid droplets are present in all eukaryotic cells and are discernible as round intracellular structures. They serve to store metabolic energy in form of neutral lipids, commonly known as 鈥渇at鈥�. This energy is stored primarily in the fatty acids that are esterified in triacylglycerols and steryl esters, and is released upon ?-oxidation of these fatty acids. Lipid droplets are thus implicated in many of the pandemic diseases such as obesity, insulin resistance, atherosclerosis and lipotoxicity. The hydrophobic core of lipid droplets is covered by a phospholipid monolayer and they harbor a specific set of proteins, many of which function in neutral lipid metabolism, such as lipases or acyltransferases. We and others have previously shown that these lipid droplets are closely associated with the membrane of the endoplasmic reticulum. This association is functionally important as it allows the transfer of integral membrane as well as that of lipids between the two compartments. The precise nature of this association between the two compartments, however, has remained elusive. Recent results from our laboratory indicate that lipid droplets are accessible to proteins from within the luminal compartment of the endoplasmic reticulum, indicating that lipid droplets may form inside this luminal compartment. Should this indeed be the case, the biogenesis of lipid droplets would be similar to that of lipoprotein particles, which in essence are a functionalized, secreted form of lipid droplets. The aim of this part of the proposal thus is to define the nature of the association between the endoplasmic reticulum and lipid droplets. Therefore, we will determine whether endoplasmic reticulum resident integral membrane proteins can laterally move to localize over lipid droplets. In addition, we will compare the structure and topology of integral membrane proteins between their localization in the endoplasmic reticulum and their lipid droplet localization. The results of this study will thus provide new essential information to define and understand how exactly proteins and lipids can be exchanged between the endoplasmic reticulum and the lipid droplet storage compartment. This information is essential for an improved understanding of the etiology of lipid-related diseases. The CAP superfamily of proteins is named after the three founding members of this family, Cysteine-rich secretory proteins (CRISP), Antigen 5, and Pathogenesis-related 1 (PR-1). CAP family members are implicated in many fundamental biological processes, ranging from immune defense in mammals and plants, sperm maturation and fertilization, pathogen virulence, venom toxicity and even prostate and brain cancer. CAP family members are mostly secreted glycoproteins that are stable in the extracellular space. The mode of action of these proteins, however, has remained elusive. We could previously show that the CAP family members in yeast, known as Pathogen Related in Yeast (Pry), bind and thereby solubilize sterols and related hydrophobic compounds. Sterol binding is a conserved feature of many CAP proteins, including human CRISP2, a CAP superfamily member that is expressed in the testis and epididymis and participates in sperm鈥揺gg interaction during fertilization, and the plant PR-1 protein, which is synthesized in response to infections of plants with pathogens. Recent evidence indicates that these proteins not only are capable of binding sterols, but they independently also can bind fatty acids. We will test whether Pry proteins bind fatty acids, define the binding site by site-directed mutagenesis and test whether lipid binding of these proteins is important for their membrane association, permeabilization, and in vivo function. These results will thus help to define the molecular mode of action of these proteins and thereby improve our understanding of the physiological function these proteins exert in both health and disease.

    Lipid Storage and Export
    Statut: Termin茅

    顿茅产耻迟 01.10.2014
    Fin 30.11.2017
    Financement FNS 
    Lipid homeostasis is essential to ensure proper membrane function and cell viability. In this proposal, we address two basic aspects of this homeostatic regulation. In the first part, the topology of lipid storage and the biogenesis of lipid droplets are being addressed. In the second part, we propose to perform a detailed structural and functional analysis of a novel class of secreted sterol-binding proteins, the Pry (pathogen related in yeast) family. Both projects build on our established expertise and they constitute a rational continuation of our previously published work in this field. Lipid droplets constitute a globular intracellular compartment that is dedicated to the storage of fat/neutral lipids. The droplet is composed of a core of neutral lipids, particularly triacylglycerols and steryl esters and covered by a monolayer membrane onto which a small number of proteins associate. Many of these proteins function in lipid metabolism, for example as acyltransferases or lipases. We have previously shown that some of these lipid droplet associated proteins are integral membrane proteins and that they can move between the membrane of the endoplasmic reticulum (ER) and the surface of lipid droplets. These as well as other observations indicate that lipid droplets are closely associated with the ER membrane. The nature of this association, however, has not yet been resolved and is currently debated. Here, we will test if lipid droplets are accessible from within the ER lumen by targeting soluble lipid droplet-associated proteins into the luminal space of the ER. Preliminary results indicate that this indeed is the case, thus challenging some of the current models of lipid droplet biogenesis. To complement these studies, we will artificially generate ER-luminal lipid droplets by targeting heterologous lipid droplet-scaffolding proteins inside the ER luminal space. We have previously shown that cytosolic expression of such proteins in yeast results in the induction of lipid droplets and preliminary results indicate that lipid droplet induction is also observed if these proteins are targeted into the ER lumen. Thus having an experimental system to form cytosolic as well as ER luminal lipid droplets will allows us to compare their properties with naturally formed lipid droplets and thus allow us to define their origin and the nature of their association with the ER. Pry proteins belong to a large protein superfamily known as CAP/SCP (sperm coating proteins). These proteins are implicated in a multitude of physiologically important processes in all kingdoms of life, including but not limited to immune defense in animals and plants, pathogen virulence, sperm maturation and fertilization, venom toxicity and prostate and brain cancer. CAP proteins are mostly secreted glycoproteins and are stable in the extracellular environments over a wide range of conditions. Their mode of action, however, has remained elusive. We could recently show that the yeast Pry proteins bind sterols and related small hydrophobic compounds in vivo and in vitro. This sterol-binding activity is confined to the CAP domain of Pry1, and conserved in a human CAP family member, CRISP2, suggesting that CAP family members exert their various functions through binding sterols or related small hydrophobic compounds. Aim of this part of the project is to characterize the ligand binding activity of Pry1 at the structural level and to define its ligand-specificity. The results of this work will provide the foundation to understand the mode of action of this important class of proteins; not only in fungi but also in plants and vertebrates.

    PM-DOMAIN Role of lipid-protein interactions in formation of plasma membrane microdomains
    Statut: Termin茅

    顿茅产耻迟 01.08.2014
    Fin 31.07.2015
    Financement SCIEX 

    Lipid Acetylation, Storage, Export and Degradation
    Statut: Termin茅

    顿茅产耻迟 01.07.2011
    Fin 30.09.2014
    Financement FNS 
    Lipid Acetylation, Storage, Export and Degradation

    Membrane topology of lipid synthesis, transport, and turnover and their role in the physiology of starvation and aging
    Statut: Termin茅

    顿茅产耻迟 01.08.2009
    Fin 28.02.2013
    Financement FNS 
    The four groups involved in this project all are engaged in basic research and utilize the yeast Saccharomyces cerevisiae as a model organism, but work on research topics that are not related at first sight. The currently studied topics relevant for this common proposal include: a) the bud neck structures preventing nuclear pores and the Sec61-translocon to laterally diffuse from the mother cell into the growing daughter cell in the nuclear envelope and cortical ER, respectively, and the role of this barrier in retaining aged and damaged cell components in the mother cell to protect daughter cells from premature aging; b) the mechanisms by which nutrient signaling pathways control - in response to the quality and concentration of nutrients 鈥� the orchestrated entry into and exit from a quiescent (G0) state; c) the regulation of lipid droplet biogenesis and the corresponding enzymes that control both storage/mobilization of essential lipids and disposal of oxysterols; d) the role of sphingo- and glycerophospholipid biosynthesis for GPI anchoring and membrane biosynthesis. New results obtained in all four groups form the basis for a set of new experimental ideas, which can be tested much more efficiently in an integrated project, which combines the expertise (including established methodologies) of the four participating groups. First, new results revealing the importance of sphingolipids in maintaining the bud-neck diffusion barrier for protein complexes in the ER membrane, raise the question of whether sphingolipids play a structural role as components of the membrane barrier, or whether they indirectly regulate the structure of this barrier by controlling signaling events. Genetic experiments will allow distinguishing between these possibilities. Also, the ability of the barrier to retain single span proteins and lipids will have to be tested. Second, the recent finding that the maintenance of the cortical ER barrier depends on Rim15p, a key downstream effector of various nutrient-sensitive protein kinases (such as the protein kinase A [PKA] and target of rapamycin complex 1 [TORC1]), raises the question of whether and how nutrient signaling impinges on the establishment of a proper cortical ER barrier. Third, newly made observations indicate that enzymes preparing lipids for storage are induced and activated in cells entering G0, whereas the lipases hydrolyzing ergosterol esters and triacylglycerols are activated in cells exiting G0 (upon refeeding), suggesting that the nutrient signaling pathways, which control entry into and exit from G0 (e.g., PKA and TORC1 pathways), may also regulate the storage and mobilization of lipids. Data also raise the question of whether lipid droplets are directed towards the incipient bud and segregated into growing daughter cells following resumption of growth of cells that exit G0. Fourthly, it recently became clear that essential acyltransferases of glycerophospholipid-biosynthesis have their active sites in the ER lumen, that also ergosterol and diacylglycerol are acylated for storage in lipid droplets by acyltransferases, which have their active site on the luminal side of the ER membrane, and that toxic lipids are acetylated in the lumen of the ER. The luminal localization of the catalytic sites of various acyltransferases in turn implies that substrates for these acyltransferases such as ergosterol, glycerol-3- phosphate, or acyl-CoA will have to be either flopped from the cytosol across the ER membrane, or to be synthesized in the ER-lumen. Flopping may be mediated by dedicated floppases/scramblases or else be an integral mechanism of the acyltransferases, which are all integral membrane proteins. Finally, our data also raise questions regarding the biogenesis of lipid droplets, specifically how enzymes controlling neutral lipid metabolism are regulated. We will make ample use of recent advances in confocal fluorescence microscopy, synthetic genetic array and mass spectrometric methods.

    Speicherung, Mobilisierung und Umsatz von Neutrallipiden
    Statut: Termin茅

    顿茅产耻迟 01.07.2008
    Fin 30.06.2011
    Financement FNS 
    All organisms store excess of metabolic energy as neutral lipids or 鈥渇at鈥�. Neutral lipids are composed of triacylglycerol (TAG) and, in eukaryotes, also contain steryl esters (STE). The fatty acids present in TAG and STE provide at least ten-times as much energy as that of an equal mass of hydrated carbohydrates or proteins. This energy is typically released by degradation through beta-oxidation of the long chain fatty acids present in TAG and STE, either in mitochondria (animal and plant cells) or in peroxisomes (yeasts). Because these neutral lipids are extremely hydrophobic, they are stored in intracellular lipid droplets (LDs), organelles that are dedicated to neutral lipid storage. Multicellular organisms have differentiated cell types such as adipocytes that are specialized for storage of neutral lipids in the form of giant cytosolic LDs, but have also developed pathways to secret neutral lipid in form of lipoproteins and milk lipid globules. Intracellular LDs are surrounded by an unusual lipid monolayer membrane and harbor only a limited number of different proteins, many of which are directly implicated in lipid metabolism. How precisely these LDs are formed and how their biogenesis is regulated, however, is poorly understood and will be addressed in the present research program. The second main question that will be addressed is how access, turnover, and degradation of this neutral lipid pool are governed. We and others have identified lipases that are required for the degradation of TAG and STE, and many of these lipases localize to LDs. How these lipases are activated and how they gain access to their insoluble substrates, however, remains to be defined. Understanding the regulation and turnover of neutral lipids is important, as it will help to define some of the problems associated with the metabolic syndrome, obesity, type 2 diabetes, and its associated cardiovascular problems, all of which are associated with an excess accumulation of neutral lipids and elevated levels of circulating free fatty acids, which ultimately results in an inflammatory response and insulin resistance. Our studies will be performed mainly in the unicellular eukaryote yeast, as a genetically tractable model, because it allows unprecedented experimental control over LD biogenesis and neutral lipid degradation. Employing a strain with which we can induce biogenesis as well as turnover of LDs, we will characterize the biogenetic- and degradative-intermediates morphologically by confocal as well as electron microscopy; determine the topology of the two TAG synthesizing enzymes, Lro1p and Dga1p, to define whether TAG synthesis is defined to the lumenal domain of the ER; and employ genetic screens to identify mutations that block LD biogenesis or turnover. We have recently identified protein kinases that are important for the mobilization of TAG, STE, and for the phosphorylation of a key lipase in STE turnover, Yeh2p. The role of these kinases in neutral lipid turnover will be characterized in more detail and their target will be defined. Neutral lipids in the ER have recently been suggested to act in retrotranslocation of misfolded proteins, a hypothesis that will be tested using yeast mutants that lack neutral lipids. Accumulation of neutral lipids in the ER may induce ER stress and insulin resistance in mammalian cells. A possible causative relation between TAG accumulation and ER stress will be investigated using yeast strains that accumulate TAG. We have recently characterized a novel neutral lipid modification, the reversible acetylation and deacetylation of sterols. We propose that this acetylation cycle acts to proofread the structure of sterols and possibly other hydrophobic compounds to ensure that only properly synthesized sterols are incorporated into cellular membranes, as acetylation acts as a signal for the secretion of the acetylated compound.

    Flow cytometry opens new avenues for cell-based analyses in biomedical research
    Statut: Termin茅

    顿茅产耻迟 01.08.2007
    Fin 31.07.2008
    Financement FNS 
    This research proposal requests funding for the acquisition of a flow cytometer equipped with a temperature-controlled autosampler. Such an instrument allows a quantitative readout from a variety of fluorescent cell-based assay systems, which are being used, for example, to analyze cell cycle progression, to monitor apoptosis to quantify lipid transport, and to analyze cell surface expression of marker proteins. In combination with an autosampler, the instrument is suited for high-throughput screens, which have now become extremely successful for gene discovery in organisms for which deletion libraries or siRNA collections are available, such as yeast, C. elegans, T. brucei, and mammalian cells. There is presently no flow cytometer or equivalent instrument available at our campus. The instrument would thus allow us to perform experiments that are currently either not possible, or require much more time and reagents than a corresponding readout based on flow cytometry. The four SNF-funded research groups applying for this grant will be the main users of this instrument, but additional groups have already expressed their interest in using this equipment. The four groups will employ the instrument to study (i) lipid homeostasis in yeast, (ii) the connection between the circadian clock and aging, (iii) the coordination between mitochondrial biogenesis and the cell cycle in the parasitic protozoan, Trypanosoma brucei, and (iv) the role of adipose tissues of different origins in obesity-associated vascular dysfunctions. As is evident from this list of topics, the requested instrument is extremely versatile and thus will serve the needs and will be open to the more than 25 research groups in the Life Sciences that are located on the campus. This flow cytometer would thus well complement more qualitative readouts that are presently typically performed by fluorescent microscopy.

    Lipid homeostasis in yeast
    Statut: Termin茅

    顿茅产耻迟 01.07.2006
    Fin 30.06.2008
    Financement FNS 

    Eukaryotic sterol homeostasis: Sterol acetylation/deacetylation cycle in Saccharomyces cerevisiae
    Statut: Termin茅

    顿茅产耻迟 01.10.2003
    Fin 26.05.2009
    Financement Unifr 

    Eukaryotic sterol homeostasis: Steryl ester hydrolases in Saccharomyces cerevisiae
    Statut: Termin茅

    顿茅产耻迟 01.10.2002
    Fin 29.09.2006
    Financement Unifr 
    http://ethesis.unifr.ch/theses/downloads.php?file=KoeffelR.pdf

    Lipid homeostasis in yeast
    Statut: Termin茅

    顿茅产耻迟 01.07.2002
    Fin 30.06.2006
    Financement FNS 
    Aim of the present project is to characterize the molecular mechanisms that establish and maintain lipid homeostasis, employing Saccharomyces cerevisiae as a genetically tractable eukaryotic model organism. Homeostatic control of the lipid composition of cellular membranes ensures membrane function despite variations in extra- and intra-cellular conditions. Membrane function, in this definition, covers more than the established role of a membrane to serve as permeability barrier, but also includes less well-characterized functions of membranes that are important for maintaining compartmental identity, for protein sorting, and for signal transduction. These vital cellular processes are thus likely to be modulated by the lipid composition of intracellular membranes. Despite the increasingly recognized functional role of lipids in various cellular functions, our understanding of how the lipid composition of a membrane is regulated and how this regulation is integrated with other cellular processes is only poorly understood.

    MICRODOMAINS: Microdomains
    Statut: Termin茅

    顿茅产耻迟 19.05.2000
    Fin 31.05.2003
    Financement Europe 

    Mechanisms of Bax targeting and translocation
    Statut: Termin茅

    顿茅产耻迟 01.10.1999
    Fin 07.11.2003
    Financement Unifr 

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