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Graz University of Technology

Austria

Institute of Biochemistry

Annual Report 2010

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Staff Members of the Institute of Biochemistry, TU Graz

http://www.biochemistry.tugraz.at/

Professors

Peter Macheroux (Full Professor & Head of the Institute) peter.macheroux@tugraz.at; Tel.: +43-(0)316-873-6450

Günther Daum (Associate Professor)

guenther.daum@tugraz.at; Tel.: +43-(0)316-873-6462

Albin Hermetter (Associate Professor) albin.hermetter@tugraz.at; Tel.: +43-(0)316-873-6457

Michael Murkovic (Associate Professor)

michael.murkovic@tugraz.at; Tel.: +43-(0)316-873-6495

Karin Athenstaedt (Independent Group Leader) karin.athenstaedt@tugraz.at; Tel.: +43-(0)316-873-6460

Assistants

Ines Waldner-Scott ines.waldner-scott@tugraz.at; Tel.: +43-(0)316-873-6454

Tanja Knaus

tanja.knaus@tugraz.at; Tel.: +43-(0)316-873-6463

Alexandra Binter alexandra.binter@tugraz.at; Tel.: +43-(0)316-873-6453

Silvia Wallner

silvia.wallner@tugraz.at; Tel.: +43-(0)316-873-6955

Office

Annemarie Portschy portschy@tugraz.at; Tel.: +43-(0)316-873-6451; Fax: +43-(0)316-873-6952

Technical Staff

Claudia Hrastnik; claudia.hrastnik@tugraz.at; Tel.: +43-(0)316-873-6460

Steve Stipsits; steve.stipsits@tugraz.at; Tel.: +43-(0)316-873-6464 Rosemarie Trenker-El-Toukhy; r.trenker-el-toukhy@tugraz.at; Tel.: +43-(0)316-873-6464

Elfriede Zenzmaier; elfriede.zenzmaier@tugraz.at; Tel.: +43-(0)316-873-6467 Alma Ljubijankic; alma.ljubijankic@tugraz.at; Tel.: +43-(0)316-873-6460 or 6498

Eva Maria Pointner; eva.pointner@tugraz.at; Tel.: +43-(0)316-873-6453

Leo Hofer (Workshop); leo.hofer@tugraz.at; Tel.: +43-(0)316-873-8431 or 8433

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Brief History of the Institute of Biochemistry The Institute of Biochemistry and Food Chemistry was born out of the division of the Institute

of Biochemical Technology, Food Chemistry and Microchemistry of the former School of

Technology Graz. Together with all the other chemistry institutes, it was located in the old Chemistry Building on Baron Mandell's ground, corner Technikerstrasse-Mandellstrasse. 1929 The Institute of Technical Biochemistry and Microbiology moved to the building of

the Fürstlich-Dietrichstein-Stiftung, Schlögelgasse 9, in which all the biosciences were then concentrated.

1945 G. GORBACH - initially in the rank of a docent and soon thereafter as a.o. Professor -

took over to lead the institute. The institute was renamed Institute of Biochemical

Technology and Food Chemistry. 1948 G. GORBACH was nominated full professor and head of the institute. In succession of

the famous Graz School of Microchemistry founded by Prof. F. PREGL and Prof. F. EMICH, Prof. GORBACH was one of the most prominent and active leaders in the fields of microchemistry, microbiology and nutritional sciences. After World War II, questions of water quality and waste water disposal became urgent; hence, the group of Prof. K. STUNDL, which at that time was part of the institute, was gaining importance. In addition, a division to fight dry-rot supervised by Dr. KUNZE and after his demise by H. SALOMON, was also affiliated with the institute.

1955 In honour of the founder of microchemistry and former professor at the Graz

University of Technology, the extended laboratory was called EMICH-Laboratories. At the same time, the institute was renamed Institute of Biochemical Technology, Food

Chemistry and Microchemistry. Lectures and laboratory courses were held in biochemistry, biochemical technology, food chemistry and food technology, technical microscopy and microchemistry. In addition, the institute covered technical microbiology together with biological and bacteriological analysis - with the exception of pathogenic microorganisms - and a lecture in organic raw materials sciences. 1970 After the decease of Prof. GORBACH, Prof. GRUBITSCH was appointed head of the

institute. Towards the end of the sixties, the division for water and waste water disposal headed by Prof. STUNDL was drawn out of the institute and established as an independent institute. Prof. SPITZY was nominated professor of general chemistry, micro- and radiochemistry. This division was also drawn out of the mother institute and at the end of the sixties moved to a new building.

1973 Division of the Institute for Biochemical Technology, Food Technology and

Microchemistry took place. At first, biochemical technology together with food technology formed a new institute now called Institute of Biotechnology and Food

Chemistry headed by Prof. LAFFERTY. 1973 Dr. F. PALTAUF, docent at the Karl-Franzens-University Graz, was appointed

professor and head of the newly established Institute of Biochemistry. The interest of Prof. PALTAUF in studying biological membranes and lipids laid the foundation for the future direction of research. G. DAUM, S. D. KOHLWEIN, and A. HERMETTER

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joined the institute. All three young scientists were given the chance to work as post docs in renown laboratories in Switzerland and the USA: G. DAUM with the groups of G. Schatz (Basel, Switzerland) and R. Schekman (Berkeley, USA), A. HERMETTER with J. R. Lakowicz (Baltimore, USA) and S. D. KOHLWEIN with S. A. Henry (New York, USA). Consequently, independent research groups specialized in cell biology (G. D.), biophysics (A. H.) and molecular biology (S. D. K.) evolved at the institute in Graz, with the group of Prof. F. PALTAUF still focusing on the chemistry and biochemistry of lipids.

Teaching was always a major task of the institute. Lectures, seminars and laboratory courses in basic biochemistry were complemented by special lectures, seminars, and courses held by the assistants who became docents in 1985 (G. D.), 1987 (A. H.), and 1992 (S. D. K.). Lectures in food chemistry and technology were held by C. WEBER and H. SALOMON. Hence the institute was renamed Institute of Biochemistry and Food Chemistry. 1990 The institute moved to a new building at Petersgasse 12. The move was accompanied

by the expansion of individual research groups and the acquisition of new equipment essential for the participation in novel collaborative efforts at the national and international level. Thus, the Institute of Biochemistry, together with partner institutes from the Karl-Franzens University was the driving force to establish Graz as a centre of competence in lipid research.

1993 W. PFANNHAUSER was appointed as professor of food chemistry. Through his own

enthusiasm and engagement and that of his collaborators, this new section of the institute rapidly developed and offered students additional opportunities to receive a timely education.

2000 The two sections, biochemistry and food chemistry, being independent of each other

with respect to personnel, teaching, and research, were separated into the Institute of

Biochemistry (Head Prof. PALTAUF) and the new Institute of Food Chemistry and

Technology (Head Prof. PFANNHAUSER). 2001 After F. PALTAUF’s retirement, in September 2001, G. DAUM was elected head of

the institute. S. D. KOHLWEIN was appointed full professor of biochemistry at the Karl-Franzens University Graz.

2003 P. MACHEROUX was appointed full professor of biochemistry in September 2003

and head of the Institute of Biochemistry in January 2004. His research interests revolve around topics in protein biochemistry and enzymology and shall strengthen the already existing activities in this area.

2007 K. ATHENSTAEDT, a long-time associate of Prof. DAUM, received the venia

legendi for biochemistry. Dr. Athenstaedt was the first woman to complete the traditional habilitation at the Institute of Biochemistry.

2009 After the retirement of Prof. PFANNHAUSER in 2008, the Institute of Food

Chemistry and Technology was disbanded and the research group of Prof. M. MURKOVIC joined the Institute of Biochemistry increasing the number of independent research groups to five.

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Highlights of 2010 In 2010, Peter Macheroux took a break from his teaching duties and spent most of the year from mid-February until the end of July on sabbatical leave in the Dept. of Chemistry & Chemical Biology at Cornell University (hosted by Prof. Steve Ealick). This was a truly unique experience not just because of the opportunity to acquire insight into new scientific methods but also in terms of getting to know new colleagues and customs, such as commencement day where students obtain their academic degree during a traditional celebration. In spring 2010 the Austrian Science Fund (FWF) approved a new research project to investigate the interaction of quinone reductase with the proteasome that controls the localization of some transcription factors (Project P22361: “Mechanism of redox controlled protein degradation”). Finally, Dr. Andreas Winkler, a former PhD student of the PhD program “Molecular Enzymology” and currently a postdoctoral fellow at the Max-Planck Institute of Molecular Medicine in Heidelberg received the dissertation award 2010 of the Austrian Society for Molecular Biosciences and Biotechnology (ÖGMBT) for the thesis he has completed in our institute the year before.

Impressions from the sabbatical of P. Macheroux

Commencement day at Cornell in May 2010

Karlheinz Grillitsch was the 100th student to receive his PhD at the Institute of Biochemistry. Opponents were K. Lohner (left) and G. Daum (right of the candidate); chairman A. Hermetter (right).

In the group of Günther Daum, three new projects were started in 2010. Whereas the FWF project P23029 is devoted to the fundamental investigation of yeast lipases, the Translational Research Project TRP009 addresses applied aspects of Pichia lipidomics. Finally, the lab of Günther Daum is involved in investigations of protein expression in the framework of the Austrian Centre of Industrial Biotechnology (ACIB). A highlight in graduate student education was the completion of the Doctoral Thesis of Karlheinz Grillitsch because this was the 100th PhD degree received at the Institute of Biochemistry, TU Graz. In 2010, Günther Daum started his three year term as President of the International Conference on the Bioscience of Lipids (ICBL). In May 2010, he was Organizer and Chairman of the FEBS Workshop Microbial Lipids: From Genomics to Lipidomics, in Vienna, Austria. This was the first conference of this type, but due to its success with more than 100 participants the next meeting has already be planned for 2012 in Bern, Switzerland.

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In 2010 Karin Athenstaedt continued her work in our institute as an independent researcher. With her project she set the cornerstone to establish her own research group. Karin Athenstaedt’s work is devoted to lipid metabolism in yeast. Basic research in Albin Hermetter’s group was performed in three projects focusing on lipid (patho)physiology. His contribution to the GOLD project (speaker R. Zechner, ZMB, University of Graz, funded by the GEN-AU program of the BM.W_F) is devoted to the functional proteomic analysis of (phospho)lipases relevant to lipid-associated disorders. In the SFB LIPOTOX (FWF project, speaker R. Zechner) and the EuroMEMBRANE consortium OXPHOS (speaker P. Kinnunen, Aalto University, Helsinki), A. Hermetter studies the toxicity of oxidized phospholipids in the cells of the arterial wall (funded by FWF). Research in this field is also supported by the PhD program “Molecular Enzymology” (FWF). Ute Stemmer who is a PhD student in the SFB LIPOTOX was awarded the prize for the best oral presentation by young researchers entitled “Oxidized phospholipids: uptake and targeting in RAW 264.7 macrophages” at the International Conference on the Bioscience of Lipids in Bilbao, Spain, September 7 – 11, 2010.

K. Athenstaedt

M. Murkovic

Ute Stemmer (in the center) received the BBA Award presented by W. Dowhan (left; BBA Executive Editor) for the best oral presentation by young researchers at the International Conference on the Bioscience of Lipids in Bilbao, Spain, September 7 – 11, 2010.

In 2010, Alan Zeb finished his PhD Thesis in the group “Chemistry of Functional Foods” directed by Michael Murkovic in which the interaction of carotenoids with triglycerides from edible oils was characterized in detail. The pro- and antioxidant action of carotenoids was evaluated by a detailed analysis of the oxidation products by LC-MS. Several oxidation products of triglycerides were newly identified. Four manuscripts were published on this topic. In 2010 the biannual conference of the Austrian Food Chemists was held at Schloss Seggau near Leibnitz. At this conference around 100 participants from Austria and the neighboring countries discussed the issue of food quality on the molecular level.

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Biochemistry group

Group leader: Peter Macheroux Secretary: Annemarie Portschy Postdoctoral Fellow: Ines Waldner-Scott PhD students: Thomas Bergner, Alexandra Binter, Venugopal Gudipati, Tanja Knaus, Wolf-Dieter Lienhart, Silvia Wallner Technicians: Eva Maria Pointner, Steve Stipsits, Rosemarie Trenker-El-Toukhy Alumni 2010: Katrin Fantur (PhD) General description The fundamental questions in the study of enzymes, the bio-catalysts of all living organisms, revolve around their ability to select a substrate (substrate specificity) and subject this substrate to a predetermined chemical reaction (reaction and regio-specificity). In general, only a few amino acid residues in the "active site" of an enzyme are involved in this process and hence provide the key to the processes taking place during enzyme catalysis. Therefore, the focus of our research is to achieve a deeper understanding of the functional role of amino acids in the active site of enzymes with regard to substrate-recognition and stereo- and regiospecificity of the chemical transformation. In addition, we are also interested in substrate-triggered conformational changes and how enzymes utilize cofactors (flavin, nicotinamide) to achieve catalysis. Towards these aims we employ a multidisciplinary approach encompassing kinetic, thermodynamic, spectroscopic and structural techniques. In addition, we use site-directed mutagenesis to generate mutant enzymes to probe their functional role in the mentioned processes. Furthermore, we collaborate with our partners in academia and industry to develop inhibitors for enzymes, which can yield important new insights into enzyme mechanisms and can be useful as potential lead compounds in the design of new drugs. The methods established in our laboratory comprise kinetic (stopped-flow and rapid quench analysis of enzymatic reactions), thermodynamic (isothermal titration microcalorimetry) and spectroscopic (fluorescence, circular dichroism and UV/VIS absorbance) methods. We also frequently use MALDI-TOF and ESI mass spectrometry, protein purification techniques (chromatography and electrophoresis) and modern molecular biology methods to clone and express genes of interest. A brief description of our current research projects is given below. Berberine bridge enzyme & other flavin-dependent plant oxidases

Berberine bridge enzyme (BBE) is a central enzyme in the biosynthesis of berberine, a pharmaceutically important alkaloid. The enzyme possesses a covalently attached FAD moiety, which is essential for catalysis. The reaction involves the oxidation of the N-methyl group of the substrate (S)-reticuline by the enzyme-bound flavin and concomitant formation of a carbon-carbon bond (the “bridge”). The ultimate acceptor of the substrate-derived electrons is dioxygen, which reoxidizes the flavin to its resting state:

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The BBE-catalysed oxidative carbon-carbon bond formation is a new example of the versatility of the flavin cofactor in biochemical reactions. Our goal is to understand the oxidative cyclization reaction by a biochemical and structural approach. We have developed a new expression system for BBE (using cDNA from Eschscholzia

california, gold poppy) in Pichia pastoris, which produces large amounts of the protein (ca. 500 mg from a 10-L culture). The availability of suitable quantities of BBE enabled us to crystallize the protein and to solve the structure in collaboration with Prof. Karl Gruber at the Karl-Franzens University Graz (see below).

Based on the three-dimensional structure of BBE, we have performed a site-directed mutagenesis program to investigate the role of amino acids present in the active site of the enzyme. In conjunction with other experiments, this has led to the formulation of a new reaction mechanism for the enzyme (thesis project of Andreas Winkler). Currently, Silvia Wallner investigates alternative covalent modifications in the 8α-position in BBE variants that contain either aspartate or tyrosine instead of a histidine. In collaboration with Prof. Toni Kutchan at the Donald Danforth Plant Science Center in St. Louis, we have identified other

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plant genes that apparently encode flavin-dependent oxidases. These genes are currently expressed in our laboratory in order to characterize the role of the enzymes in alkaloid biosynthesis (thesis project of Silvia Wallner).

Dipeptidylpeptidase III

Dipeptidyl-peptidases III (DPPIII; EC 3.4.14.4) are Zn-dependent enzymes with molecular masses of ca. 80-85 kDa that specifically cleave the first two amino acids from the N-terminus of different length peptides. All known DPPIII sequences contain the unique motif HEXXGH, which enabled the recognition of the dipeptidyl-peptidase III family as a distinct evolutionary metallopeptidase family (M49). In mammals, DPPIII is associated with important physiological functions such as pain regulation, and hence the enzyme is a potential drug target. Previously, Sigrid Deller and Nina Jajcanin-Jozic have successfully expressed, purified and characterized the recombinant yeast enzyme, and Pravas Baral in Karl Gruber’s laboratory at the Karl-Franzens-University has elucidated the crystal structure of the yeast protein. This work revealed that yeast DPPIII features a novel protein topology.

Structure of human DPPIII in its open form (right) and peptide liganded (closed) form

In collaboration with a structural genomics group in Toronto led by Dr. Sirano Dhe-Paganon, Gustavo Arruda has solved the structure of the human enzyme both in its open (right, above) and closed conformation (left, above). The latter structure was obtained by co-crystallization of a tightly binding peptide to an inactive variant of human DPPIII. These two new structures constitute a major breakthrough in our effort to understand the physiological role of the enzyme and pave the way for the development of potentially useful inhibitors of the enzyme (Gustavo Arruda’s thesis project in Prof. Gruber’s laboratory supported by Alexandra Binter).

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Luciferase and LuxF

The emission of light by biological species (bioluminescence) is a fascinating process found in diverse organisms such as bacteria, funghi, insects, fish, limpets and nematodes. In all cases the bioluminescent process is based on a chemiluminescent reaction in which the chemical energy is (partially) transformed into light energy ("cold light"). All bioluminescent processes require a luciferase, i.e. an enzyme catalyzing the chemiluminescent reaction, and a luciferin, which can be considered a coenzyme. During the bioluminescent reaction the luciferin is generated in an excited state and serves as the emitter of light energy. In our laboratory, we are interested in the bioluminescence of marine photobacteria. In these bacteria, luciferase is composed of an alpha/beta-heterodimeric protein, which binds reduced flavinmononucleotide (FMN) as the luciferin. The reduced FMN reacts with molecular dioxygen to a hydroperoxide intermediate with subsequent oxidation of a long-chain fatty aldehyde (e.g. tetradecanal) to the corresponding fatty acid (e.g. myristic acid). During this oxidation process, an excited flavin intermediate is generated which emits light. Some marine photobacteria possess an additional protein called LuxF which was found in complex with a myristylated flavin derivative where the C-3 atom of myristic acid is covalently attached to the 6-position of the flavin ring system. It was postulated that this flavin adduct is generated in the luciferase catalyzed bioluminescent reaction. Furthermore, it was speculated that LuxF sequesters the myristylated flavin adduct in order to prevent inhibition of the bioluminescent reaction. However, both hypotheses have not been tested on a biochemical or physiological level yet. Hence, in this study we will design and perform experiments to examine the putative generation of myristylated FMN through the luciferase reaction (thesis project of Thomas Bergner supported by Steve Stipsits)

Structure of a LuxF dimer in the absence (red) and presence (blue) of the myristylated flavin derivative (pdb code 1NFP)

Nikkomycin biosynthesis

Nikkomycins are produced by several species of Streptomyces and exhibit fungicidal, insecticidal and acaricidal properties due to their strong inhibition of chitin synthase. Nikkomycins are promising compounds in the cure of the immunosuppressed, such as AIDS patients, organ transplant recipients and cancer patients undergoing chemotherapy. Nikkomycin Z (R1= uracil & R2= OH, see below) is currently in clinical trial for its antifungal activity. Structurally, nikkomycins can be classified as peptidyl nucleosides containing two

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unusual amino acids, i.e. hydroxypyridylhomothreonine (HPHT) and aminohexuronic acid with an N-glycosidically linked base:

Although the chemical structures of nikkomycins have been known since the 1970s, only a few biosynthetic steps have been investigated in detail. The steps leading to the synthesis of aminohexuronic acid are unclear. Originally, it was hypothesized that the aminohexuronic acid moiety is generated by addition of an enolpyruvyl moiety from phosphoenolpyruvate (PEP) to either the uridine or the 4-formyl-4-imidazolin-2-one analog at the 5’-position of ribose. This step is then followed by rather speculative modifications to yield the aminohexuronic acid precursor. In contrast to this hypothesis, we could recently demonstrate that UMP rather than uridine serves as the acceptor for the enolpyruvyl moiety, a reaction catalyzed by an enzyme encoded by a gene of the nikkomycin operon termed nikO. Furthermore, we could demonstrate that it is attached to the 3’- rather than the 5’-position of UMP. These results are very intriguing since none of the nikkomycins synthesized possess an enolpyruvyl group in this position of the sugar moiety. Hence, it must be concluded that the resulting 3’-enolpyruvyl-UMP is subject to rearrangement reactions where the enolpyruvyl is detached from its 3’-position and transferred to the 5’-position of the ensuing aminohexuronic acid moiety. Co-crystallization of NikO with fosfomycin yielded rod – like crystals diffracting up to 2.5 Å. A synchrotron dataset was measured at the Swiss Light Source and the structure was solved by molecular replacement using UDP-N-acetylglucosamine enolpyruvyl transferase (PDB code: 2rl1) as a model. Two molecules were found in the asymmetric unit exhibiting an inverse α,β-barrel fold with helices forming the tightly packed core and sheets shielding the hydrophobic core from the solvent:

Eech chain is comprised of two inverse α,β-barrel subunits, which are connected by a hinge region. The final structure was refined to final R/Rfree values of 17% and 19%, respectively. Crystallization trials of NikO in the presence of its product, 3’-EP-UMP, are currently under way.

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The genes that are co-transcribed with nikO have been reported and are designated as nikI,

nikJ, nikK, nikL, nikM and nikN. In order to investigate the role of the encoded proteins in the biosynthesis of the aminohexuronic acid moiety, these genes are cloned, expressed and the proteins purified for biochemical characterization and crystallization (thesis project of Alexandra Binter and Gustav Oberdorfer in Prof. Gruber’s laboratory).

Lot6p – a redox regulated switch of the proteasome

During our previous studies of bacterial quinone reductases, we have also investigated the biochemical properties of the yeast homolog Lot6p. Despite the availability of a three-dimensional structure for Lot6p (1T0I), the physiological role of the enzyme was unclear. Our recent studies have now demonstrated that the enzyme rapidly reduces quinones at the expense of a reduced nicotinamide cofactor, either NADH or NADPH. In order to further characterize the cellular role of Lot6p, we have carried out pull-down assays and identified the 20S core particle of the yeast proteasome as interaction partner. Further studies revealed that this complex recruits Yap4p, a member of the b-Zip transcription factor family, but only when the flavin-cofactor of Lot6p is in its reduced state. Oxidation of the flavin leads to dissociation of the transcription factor and relocalization to the nucleus (see scheme below).

A similar system is known from mammalian cells, where a homologous quinone reductase (NQO1) binds to the 20S proteasome and recruits important tumor suppressor proteins such as p53 and p73α. Hence, the discovery of a homologous protein interaction in yeast provides an interesting model system to investigate the molecular basis for protein complex formation and regulation of proteasomal degradation of transcription factors (postdoctoral project of Ines Waldner-Scott; thesis project of Wolf-Dieter Lienhart and Venugopal Gudipati).

Zn-dependent Alkylsulfatases Hydrolysis of alkylsulfates is an important pathway for soil and other bacteria to mobilize sulfur. Three classes of sulfatases - divided according to their reaction mechanism - have been discovered, and the recently elucidated structure of SdsA1 from Pseudomonas aeruginosa is another example of the widely occurring family of metallo-ß-lactamases. This group of alkylsulfatases is characterized by two Zn2+ atoms in the active site which activate a water molecule for nucleophilic attack on the sulfate group. SdsA1 mainly cleaves long-chain primary alkylsulfates (preferred substrate is dodecylsulfate) by stereoinversion. In other

α�

β�

β α

Lot6p Oxidation by e.g. quinones

α�

β�

β α

Lot6p

Very slow with oxygen O2 nucleus

Yap4p Yap4p Yap4p

reduced oxidized

oxygen

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words, the hydroxyl group attacks the carbon atom in the course of the reaction. Recently, a novel enzyme could be identified in Pseudomonas DSM 6611 (termed PISA1 = Pseudomonas inverting alkylsulfatase 1) which mainly cleaves secondary alkylsulfates, for example 2-octylsulfate exhibiting stereopreference for the (R)-stereoisomer. In contrast to the majority of hydrolases, which do not alter the stereochemistry of the substrate during catalysis, PISA1 is an attractive enzyme for the deracemisation of sec-alcohols. Analysis of the crystal structures of PISA1 and SdsA1 showed that the overall structure of both proteins is virtually identical and both enzymes largely share the same active-site architecture, such as a sulfate binding site (composed of two Arg), a nucleophile site composed of a binuclear Zn2+-cluster typical for metallo-ß-lactamases and an Asn/Thr-hydrogen binding network for substrate positioning. However, the active site of PISA1 features several conspicuous amino acid exchanges (see figure below: in blue active side residues in SdsA1 and green those in PISA1). These amino acids are now subject of an extensive mutagenesis program to define their role in governing substrate preference of the reaction. This project is a close collaboration with Profs. Faber (biocatalysis) and Wagner (structure determination) from the University of Graz (thesis project of Tanja Knaus in our laboratory and Markus Schober in Prof. Faber’s laboratory). Doctoral thesis completed Katrin Fantur: Friend or Foe: Iminosugars as inhibitors and pharmacological chaperones of the human lysosomal acid β-galactosidase GM1-gangliosidosis (GM1) and Morquio B disease (MBD) are rare, hereditary lysosomal storage disorders caused by mutations in the gene GLB1. Its main gene product, human lysosomal acid β-galactosidase (β-Gal) degrades N-linked oligosaccharides present in glycoproteins, GM1-gangliosides in the brain, and keratan sulfate in connective tissues. While GM1 is a phenotypically heterogenous neurodegenerative disorder, MBD is a systemic bone disease without effects on the central nervous system. Some mutations in the GLB1 gene produce stable β-Gal precursors, normally transported and processed to mature, intralysosomal β-Gal, while others affect precursor stability and intracellular transport resulting in premature protein degradation. Several misfolded enzymes were shown to be

Phe/Gly Tyr/His

Tyr/Ser

Leu/Pro

Met/Ser

Ala/Ile

Met/Ser

Tyr/Ser

Ala/Ile

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sensitive to stabilization by iminosugars, which bind at the active site to provide the proper conformation. Thus the stabilized protein may escape from degradation processes, and reach the lysosomes in an active state, as proposed for enzyme enhancement therapy (EET).

In this work the influence of novel derivatives of 1-deoxygalactonojirimycin (DGJ) on the β-Gal activity of cultured GM1 and MBD skin fibroblasts was examined. Furthermore, the effect of selected compounds on natural substrate degradation in GM1 and MBD cells was determined. Several novel iminosugars acting as pharmacological chaperones of β-Gal in specific GM1 fibroblasts were discovered and described in this work. One specific compound, DLHex-DGJ, proved to be a potent competitive inhibitor of β-Gal in vitro, and this work describes its effects on activity, protein expression, maturation and intracellular transport in

vivo in 13 fibroblast lines with GLB1 mutations. DLHex-DGJ significantly increased the catalytic activity in six GM1 cell lines, and normalization of transport and lysosomal processing of β-Gal precursors was demonstrated for selected cell lines. Furthermore, DLHex-DGJ and another, similar compound successfully reduced the level of internalized radiolabeled GM1-gangliosides in a specific GM1 cell line, suggesting that reduction of stored material is possible under certain conditions.

Specific antibodies, directed against human β-Gal, were developed with the aid of previously published protocols, and novel approaches to obtain large amounts of the purified human enzyme were tested. Two novel polyclonal anti-β-Gal peptide antibodies were produced and expression of human β-Gal in E. coli cells may provide the basis for further development of antibodies directed against the human enzyme. Large parts of this thesis were carried out at the University hospital under the guidance and supervision of Prof. Pascke.

International cooperations

Maria Abramic, Ruder Boskovic Institute Zagreb, Croatia Steve Ealick, Cornell University, Ithaca, U.S.A. Toni Kutchan, Donald Danforth Plant Science Center, St. Louis, U.S.A. Shwu Liaw, National Yang-Ming University, Taipei, Taiwan Matthias Mack, Hochschule Mannheim, Germany Bruce Palfey, University of Michigan, Ann Arbor, U.S.A.

Research projects

FWF P22361: “Mechanism of redox controlled protein degradation” FWF P19858: “Enzymes of nikkomycin biosynthesis” FWF-Doktoratskolleg “Molecular Enzymology” WTZ Austria-Croatia “Structure-function relationships in metallopeptidases of the M49 family”

Publications

1) Durchschein, K., Ferreira-da Silva, B., Wallner, S., Macheroux, P., Kroutil, W., Glueck,

S. M., Faber, K.: The flavoprotein-catalyzed reduction of aliphatic nitro-compounds represents a biocatalytic equivalent to the Nef-reaction, Green Chemistry, 2010, 12:616-619.

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2) Jajcanin-Jozic, N., Deller, S., Pavkov, T., Macheroux, P., Abramic, M.: Identification of the reactive cysteine residues in yeast dipeptidyl peptidase III, Biochimie, 2010, 92:89-96.

Award

Dissertation Award of the Austrian Society for Molecular Biosciences and Biotechnology (ÖGMBT) to Andreas Winkler for his dissertation on “Structure-function studies on

berberine bridge enzyme (BBE) from the California poppy, Eschscholzia californica”. Dr. Andreas Winkler was a PhD student in the Doktoratskolleg “Molecular Enzymology” and is now a postdoctoral fellow at the Max-Planck Institute for Molecular Medicine in Heidelberg, Germany.

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Cell Biology Group Group leader: Günther Daum Postdoctoral Fellow: Karlheinz Grillitsch (since May 2010) PhD students: Melanie Connerth, Sona Rajakumari, Karlheinz Grillitsch (till March 2010),

Miroslava Spanova, Susanne Horvath, Martina Gsell, Vid V. Flis, Vasyl’ Ivashov, Lisa Klug

Master students: Brigitte Wagner, Gerald Mascher Technicians: Claudia Hrastnik, Alma Ljubijankic (since November 2010) General description Functional organelles are the basis for regulated processes within a cell. To sequester organelles from their environment, membranes are required which not only protect the interior of the organelles but also govern communication within the cell. To study biogenesis and maintenance of biological membranes and assembly of lipids into organelle membranes our laboratory makes use of the yeast as a well established experimental system. We combine biochemical, molecular and cell biological methods addressing problems of lipid metabolism, lipid depot formation and membrane biogenesis. Specific aspects studied recently in our laboratory are (i) assembly and homeostasis of phosphatidylethanolamine in yeast organelle membranes with emphasis on the role of the major phosphatidylethanolamine synthesizing enzyme, the phosphatidylserine decarboxylase 1, (ii) neutral lipid storage in lipid particles/droplets and mobilization of these depots with emphasis on the involvement of lipases and hydrolases, and (iii) characterization of organelle membranes from the industrial yeast Pichia pastoris. Phosphatidylethanolamine, a key component of yeast organelle membranes

Work from our laboratory and from other groups had shown that phosphatidylethanolamine (PE), one of the major phospholipids of yeast membranes, is highly important for cellular function and cell proliferation. PE synthesis in the yeast is accomplished by a network of reactions including (i) synthesis of phosphatidylserine (PS) in the endoplasmic reticulum, (ii) decarboxylation of PS by mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) or (iii) Psd2p in a Golgi/vacuolar compartment, (iv) the CDP-ethanolamine pathway (Kennedy pathway) in the endoplasmic reticulum, and (v) the lysophospholipid acylation route catalyzed by Ale1p and Tgl3p. To obtain more insight into biosynthesis, assembly and homeostasis of PE, single and multiple mutants bearing defects in the respective pathways can be used. Previous investigations in our laboratory were aimed at the molecular biological identification of novel components involved in PE homeostasis of the yeast Saccharomyces

cerevisiae. For this purpose, a number of genetic screenings were performed. To obtain a global view of the role of PE in the cell and to study the effects of an unbalanced PE level we subjected a psd1∆ deletion mutant and the corresponding wild type to DNA microarray analysis and examined genome-wide changes in gene expression. Comparison of the gene expression pattern of the psd1∆ mutant with the wild type led to the identification of ~50 differentially expressed genes. Grouping of these genes into functional categories revealed that PE formation by Psd1p influenced the expression of genes involved in diverse cellular pathways including transport, carbohydrate metabolism and stress response. Currently, the

17

most promising candidates of this screening are under investigation. This study will provide novel evidence for the complex network of phospholipid synthesis in the yeast. To understand cellular PE homeostasis in more detail, we also performed experiments defining traffic routes of PE within the yeast cell. In recent studies, we investigated the plasma membrane as destination for PE traffic. We employed yeast mutants bearing defects in the different pathways of PE synthesis and demonstrated that PE formed through all four pathways can be supplied to the plasma membrane. The fatty acid composition of plasma membrane phospholipids was mostly influenced by the available pool of total species synthesized, although a certain balancing effect was observed regarding the assembly of PE species. We assume that the phospholipid composition of the plasma membrane is mainly affected by the synthesis of the respective components and subject to equilibrium, and to a lesser extent affected by specific transport and assembly processes.

Import of Psd1p into mitochondria

A central aspect of this project is characterization of Psd1p regarding its molecular properties. Like most mitochondrial proteins, Psd1p is synthesized on free cytosolic ribosomes and imported into mitochondria where processing occurs. The Psd1-proenzyme contains a mitochondrial targeting sequence, an internal sorting sequence, and an alpha- and a beta-subunit which are linked through an LGST cleavage site. Cleavage at this site leads to the mature and active form of the enzyme generating a pyruvoyl group at the N-terminus of the alpha subunit. In recent studies performed in collaboration with the laboratory of Prof. N. Pfanner, Freiburg, Germany, we investigated i) the precise import route of Psd1p through the mitochondrial membranes, ii) the specific role of the LGST cleavage site on the import, assembly and maturation of the enzyme, (iii) the topology of Psd1p in the inner mitochondrial membrane; iv) the effect of mitochondrial processing peptidases on protein maturation, and v) possible complex formation of mature Psd1p. The link between PE metabolism and peroxisome proliferation is subject to another current investigation with emphasis on the role of enzymes and lipid transport routes involved. Previous studies suggested that PE formed through all four pathways (see above) and in different subcellular membranes can be supplied to peroxisomes with comparable efficiency.

TIM23

OM

IMS

IM

?

MPP

Oct1

Tom40

Tom

?

Psd1-precursor

Psd1p-mature

18

However, mechanisms involved in these translocation processes are still unclear. To address these questions, we established in vitro and in vivo assays for studying phospholipid supply to peroxisomal membranes. We constructed a strain which lacks the gene product of OPI3, the major PE methyltransferase localized to the ER, and bears an Opi3p-GFP hybrid with an SKL targeting sequence that directs the enzyme to peroxisomes. In this “reporter mutant”, the only site of phosphatidylcholine (PC) formation via methylation of PE are the peroxisomes, and the appearance of PC becomes an indicator and measure for PE translocation from the different sites of synthesis to peroxisomes. Currently permeabilized cells of the “reporter mutant” are used to characterize the PE transport to peroxisomes in some detail. This system in combination with mutations in the different PE biosynthetic pathways will allow us to investigate the different mechanisms of PE translocation between organelles involved in aminoglycerophospholipid biosynthesis. Most recently, we also studied the link between PE metabolism and neutral lipid storage. For this purpose we analyzed lipids from strains bearing defects in PE synthesis. These analyses showed that a mutant bearing a defect in the CDP-ethanolamine pathway had a decreased level of triacylglycerols (TAG). In cki1∆dpl1∆eki1∆ mutants bearing defects in the CDP-ethanolamine pathway both the cellular and the microsomal levels of PE were markedly decreased, whereas in other mutants of PE biosynthetic routes depletion of this aminoglycerophospholipid in microsomes was less pronounced. This observation is important because the TAG synthesisizing enzyme Lro1p similar to the enzymes of the CDP-ethanolamine pathway is a component of the ER. We conclude from these results that in cki1∆dpl1∆eki1∆ insufficient local supply of PE to Lro1p was a major reason for the strongly reduced TAG level. Neutral lipid storage in lipid particles and mobilization

Yeast cells have the capacity to store neutral lipids TAG and STE (steryl esters) in subcellular structures named lipid particles/droplets. Upon requirement, TAG and STE can be mobilized and serve as building blocks for membrane biosynthesis. In a long-standing project of our laboratory, we investigate the characterization of enzymatic steps which lead to the mobilization of TAG and STE depots. A major focus of our neutral lipid project was the biochemical characterization of the three yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Previous work from our laboratory had demonstrated that deletion of TGL3 encoding the major yeast TAG lipase resulted in decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids, especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG fraction. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we carried out biochemical experiments with wild type and deletion strains bearing defects in Tgl3p, Tgl4p and Tgl5p. We demonstrated that tgl mutants had a lower level of sphingolipids and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG accumulation in these mutant cells resulted in reduced amounts of phospholipids and sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected the metabolic flux of long chain fatty acids and very long chain fatty acids required for sphingolipid and glycerophospholipid synthesis. The pattern of phosphatidylcholine (PC), PE and PS molecular species was altered in tgl deletion strains underlining the important role of TAG turnover in maintenance of the pool size and remodelling of complex membrane lipids. This study shed new light on the physiological role of TAG lipases in yeast and in general.

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Even more surprising evidence was obtained when the enzymology of the three TAG lipases Tgl3p, Tgl4p and Tgl5p was studied in some detail. Motif search analysis indicated that Tgl3p and Tgl5p did not only contain the TAG lipase but also an acyltransferase motif. Interestingly, lipid analysis revealed that deletion of TGL3 resulted in a decrease and overexpression of TGL3 in an increase of glycerophospholipids. Similar results were obtained with TGL5. Therefore, we tested purified Tgl3p and Tgl5p for acyltransferase activity. Indeed, both enzymes did not only exhibit lipase activity but also catalyzed acylation of lysophosphatidylethanolamine and lysophosphatidic acid, respectively. Experiments using variants of Tgl3p created by site-directed mutagenesis clearly demonstrated that the two enzymatic activities act independently of each other. These results demonstrated that yeast Tgl3p and Tgl5p play a dual role in lipid metabolism contributing to both anabolic and catabolic processes.

Domains of Tgl3p (from Rajakumari and Daum, 2010, J. Biol. Chem. 285, 15769-15776)

In another study, we demonstrated that the yeast TAG lipase Tgl4p, the functional ortholog of adipose TAG lipase (ATGL), catalyzes multiple functions in lipid metabolism. An extended domain and motif search analysis revealed that Tgl4p bears not only a lipase consensus domain but also a conserved motif for calcium independent phospholipases A2 (PLA2). We showed that Tgl4p exhibits TAG lipase, STE hydrolase and PLA2 activities, but also catalyzes acyl-CoA dependent acylation of lysophosphatidic acid (LPA) to phosphatidic acid. Heterologous overexpression of Tgl4p in Pichia pastoris increased total phospholipid and specifically phosphatidic acid synthesis. Moreover, deletion of TGL4 in Saccharomyces

cerevisiae showed an altered pattern of PC and phosphatidic acid molecular species. Altogether, our data suggested that yeast Tgl4p functions as hydrolytic enzyme in lipid degradation, but also contributes to fatty acid channelling and phospholipid remodelling. Several years ago we identified through a mass spectrometric approach for the first time the major lipid particle proteins of Saccharomyces cerevisiae. This approach was a milestone in the field because it identified a number of new gene products and their function and provided valuable hints for processes associated with lipid particles. Recently, a more precise lipid particle proteome analysis was initiated in collaboration with M. Karas from the Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe University, Frankfurt, Germany. This proteome study was combined with a lipidomics investigation that was performed in collaboration with H. Köfeler from the Center for Medical Research, Medical University of Graz, Austria. In this study, we compared lipid particle components from cells which were

282 Patatin domain 483

1aa 910aa

28 GGGTFG 29

31 GSSAG 31

282 Patatin domain 483

1aa 910aa

28 GGGTFG 29

31 31

Lipase

P-Lipase

HIIACQ

Acyltransferase

48 49

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grown on glucose or on oleic acid. This approach identified a number of lipid particle proteins that were already known but also some novel polypeptide candidates. We also realized through this approach that there were some differences in the lipid particle proteome from cells grown on glucose or oleic acid. Finally, mass spectrometric analyses revealed marked differences in the lipidome of lipid particles from cells grown on the two different carbon sources. This study sets the stage for further investigation of protein-lipid interaction on the surface of lipid particles and provides basic evidence for the coordinated biosynthesis of lipid and protein components from lipid particles. Another important aspect of this project was to understand cell biological consequences of dysfunctions in non-polar lipid storage. For this purpose, yeast cells were cultivated on oleic acid which was assumed to provoke lipotoxic stress. We found that under these cultivation conditions TAG synthesis was enhanced creating the major pool for the excess of fatty acids, whereas surprisingly STE synthesis was strongly inhibited. We showed that this effect was not due to decreased expression of ARE2 encoding the major yeast STE synthase at the transcriptional level, but to competitive enzymatic inhibition of Are2p by free oleic acid. As a result, a triple mutant dga1∆lro1∆are1∆ARE2

+ grown on oleate did not form substantial amounts of STE and exhibited a growth phenotype similar to the dga1∆lro1∆are1∆are2∆ quadruple mutant which lacks all four acyltransferases involved in neutral lipid synthesis and consequently also lacks lipid particles. Growth of these mutants on oleate was strongly delayed and cell viability was decreased, but rescued by an adaptation process. In these strains, oleate stress caused morphological changes of intracellular membranes, altered phospholipid composition and increased formation of ethyl esters as a possible buffer for fatty acids. Another potential non-polar storage lipid is squalene. This component belongs to the group of isoprenoids and is precursor for the synthesis of sterols, steroids and ubiquinons. In a previous study we had demonstrated that squalene accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with TAG and STE. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In a recent study, we investigated the effect of squalene on biophysical properties of lipid particles and membranes and compared these results to artificial membranes. Our experiments showed that squalene lowered the order of STE shells in lipid particles. The majority of squalene, however, was localized to the center of lipid particles where it formed a soft core together with TAG. This view was confirmed with model lipid particles. In biological and artificial membranes fluorescence spectroscopy studies revealed that it is not the absolute squalene level per se, but the squalene to ergosterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membrane there is almost no additive effect of squalene. Our results demonstrated that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties. Pichia pastoris organelles and lipids

The yeast Pichia pastoris is an important experimental system for heterologous expression of proteins. Nevertheless, surprisingly little is known about organelles of this microorganism. For this reason, we started a systematic biochemical and cell biological study to establish standardized methods of Pichia pastoris organelle isolation and characterization. Recent work

21

focused on the biochemical characterization of the plasma membrane and secretory organelles from Pichia pastoris. One major aim of this project is the qualitative and quantitative analysis of phospholipids, fatty acids and sterols from organelle membranes when Pichia pastoris is grown on different carbon sources. For this purpose, methods for the isolation of Pichia

pastoris organelle fractions were established. Standardized techniques of lipid analysis including lipidome analyses are currently employed to address these problems.

Doctoral Theses completed

Sona Rajakumari: Role of yeast triacylglycerol lipases in membrane lipid metabolism Previous work from our laboratory had demonstrated that gene products of TGL3, TGL4 and TGL5 encoding the major yeast triacylglycerol (TAG) lipases are located to lipid particles. Deletion of TGL3 and TGL4 resulted in a decreased mobilization of TAG from the lipid particles and a sporulation defect. TAG stored in tgl3∆ and tgl5∆ deletion strains contains slightly increased amounts of C22:0 and C26:0 very long chain fatty acids (VLCFAs) compared to wild type. These VLCFAs are indispensable for sphingolipid biosynthesis and crucial for raft association in yeast. Moreover, deletion of these TAG lipases results in a decreased level of membrane lipid biosynthesis. In this Thesis the role of TAG lipases in membrane lipid metabolism of the yeast Saccharomyces cerevisiae was studied in some detail. First, a metabolic link between TAG lipolysis by TAG lipases and sphingolipid and phospholipid synthesis was shown. Secondly, a dual function of Tgl3p and Tgl5p as lipases and as lysophosphatidylethanolamine or lysophosphatidic acid acyltransferase, respectively, was demonstrated. We also showed that the acyltransferase but not the lipase function of Tgl3p was essential for efficient sporulation. Finally, we also characterized Tgl4p as multifunctional protein exhibiting lipase, phospholipase A2, acyl-CoA dependent LPA acyltransferase and STE hydrolase activity. Altogether, this work shows that catabolic and anabolic activities of TAG lipases play a pivotal role in maintaining lipid homeostasis in the yeast. Karlheinz Grillitsch: Lipid storage and mobilization in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae like higher eukaryotic cells (mammals and plants) and Gram-positive bacteria contains a specified organelle for lipid storage, the lipid particle (LP). Unlike other organelles, LP are covered by a phospholipid monolayer that protects its hydrophobic interior formed from densely packed non-polar lipids steryl esters (STE) and triacylglycerols (TAG). Moreover, LP contain a small but specific set of proteins. In this Thesis, storage and mobilization of yeast neutral lipids were studied. First, biochemical properties of the three STE hydolases, Tgl1p, Yeh1p and Yeh2p were investigated. Analysis of enzymatic properties revealed distinct substrate specificities of the three proteins and involvement in sterol homeostasis. Sterol homeostasis is also linked to cell polarity. We showed that two effectors of cell polarity, Ste20p and Cla4p, function as negative modulators of sterol biosynthesis. A major part of this Thesis was devoted to description of the molecular composition of the yeast LP and its modulation upon changes in cultivation conditions. For this purpose, LP from cells grown on either glucose or oleate were analyzed. Strong incorporation of the mono-unsaturated oleic acid into TAG and most phospholipids was observed upon shifting cells to oleate medium. Most notably, the balanced 1:1 ratio of TAG to STE in cells grown on glucose was strongly increased on oleate. Change of the medium also led to changes in the LP protein pattern. This was demonstrated by a combined

22

lipidomic/proteomic approach which also revealed several novel putative LP proteins. Finally, LP protein targeting and topology were studied using the squalene epoxidase Erg1p, a typical LP protein, as a model. These studies showed that the majority of the protein faced the cytosol, and only a small part was protected by the membrane. We assume that this may be a general feature of LP proteins. Melanie Connerth: Lipid traffic in the yeast The yeast Saccharomyces cerevisiae is a suitable organism for studies of lipid homeostasis in the cell. Processes involved in the biosynthesis of lipid compounds such as fatty acids, neutral lipids and phospholipids are well understood and located to different subcellular compartments. Two major yeast organelles involved in fatty acid turnover are peroxisomes harboring steps of beta-oxidation and lipid particles (LP) which are the storage organelle for excess fatty acids in the cell. Biogenesis of both compartments is not completely understood yet and little is known about mechanisms of lipid supply to either of these organelles. This Thesis was aimed at elucidation of lipid traffic routes towards and from peroxisomes and LP and the interplay of fatty acid utilization processes in both compartments. Initially, phospholipid supply to peroxisomal membranes was investigated in some detail with focus on phosphatidylethanolamine (PE) transport to peroxisomes. These studies revealed that the four different PE biosynthetic pathways contributed with different efficiency to this process. Moreover, a hybrid of the PE methyltransferase Opi3p was introduced into peroxisomes of an OPI3 deletion mutant which allowed us to measure transport of PE to peroxisomes by appearance of phosphatidylcholine (PC) in peroxisomal membranes. The major take-home message from these experiments was that direct membrane contact most likely accounts for delivery of lipids from their sites of synthesis to peroxisomes. During these studies, a link of peroxisomes with LP, the neutral lipid storage compartment, was also discovered. It was shown that exogenous oleic acid was primarily stored in LP in the form of triacylglycerols (TAG) and subsequently mobilized by a subset of already known as well as novel lipases/hydrolases for supply to peroxisomes. In the course of complete lipidome and proteome analyses of LP a novel and specific inhibitory effect of oleate on the activity of the steryl ester synthase Are2p was discovered thus revealing a new aspect of yeast lipotoxicity. In summary, data from this Thesis showed that distinct pools of fatty acids serving for different cellular processes exist in the cell, and LP play a major role in regulating lipid homeostasis. These data are a step forward in our understanding of cellular lipid traffic which may also be applicable to higher eukaryotic cells such as plants and mammals. International cooperations

N. Pfanner, Institute of Biochemistry and Molecular Biology, ZBMZ, University of Freiburg,

Germany I. Hapala, Slovak Academy of Sciences, Institute of Animal Biochemistry and Genetics,

Ivanka pri Dunaji, Slovak Republic I. Feussner, Plant Biochemistry, Albrecht-von-Haller-Institute of Plant Sciences, Georg-

August University Göttingen, Germany R. Erdmann, Institute of Physiological Chemistry, Ruhr-University Bochum, Germany M. Karas, Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe University,

Frankfurt, Germany R. Rajasekharan, Department of Biochemistry, Indian Institute of Science, Bangalore, India

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Research projects

FWF P21429: Phosphatidylserine decarboxylase FWF P18857: Neutral lipid storage and mobilization in the yeast Saccharomyces cerevisiae FWF L-178 (Translational Research): Characterization of organelles from the yeast Pichia

pastoris FWF PhD Program: Molecular Enzymology FWF P23029 Lipases of the yeast Saccharomyces cerevisiae Austrian Center of Industrial Biotechnology (ACIB): Pichia pastoris Cell factory and Protein

Production Invited Lecture

1. S. Rajakumari, M. Connerth, K. Grillitsch, S. Horvath, R. Rajasekharan and G. Daum

Fatty acid channeling from depots to biomembranes in the yeast International Lipid Symposium: Cell Biology and Metabolism, Beijing, China, 1-3 September 2010

Publications

1. Rajakumari, S. and Daum, G.

Janus-faced enzymes yeast Tgl3p and Tgl5p catalyze lipase and acyltransferase reactions Mol. Biol. Cell 21 (2010) 501-510

2. Schuiki, I., Schnabl, M., Czabany, T., Hrastnik, C. and Daum, G. Phosphatidylethanolamine synthesized by four different pathways is supplied to the plasma membrane of the yeast Saccharomyces cerevisiae. Biochim. Biophys. Acta 1801 (2010) 480-486

3. Spanova, M., Czabany, T., Zellnig, G., Leitner, E., Hapala, I. and Daum, G. Effect of lipid particle biogenesis on the subcellular distribution of squalene in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 285 (2010) 6127-6133

4. Rajakumari, S. and Daum, G. Multiple functions as lipase, steryl ester hydrolase, phospholipase and acyltransferase of Tgl4p from the yeast Saccharomyces cerevisiae. J. Biol. Chem. 285 (2010) 15769-15776

5. Wagner, M., Hoppe, K., Czabany, T., Heilmann, M., Daum, G., Feussner, I. and Fulda, M. Identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) gene from the microalga O. tauri. Plant Physiol. Biochem. 48 (2010) 407-416

6. Connerth, M., Czabany, T., Wagner, A., Zellnig, G., Leitner, E., Steyrer, E. and Daum, G. Oleate inhibits steryl ester synthesis and causes liposensitivity in the yeast. J. Biol. Chem. 285 (2010) 26832–26841

7. Rajakumari, S, Rajasekharan, R. and Daum, G. Triacylglycerol lipolysis is linked to sphingolipid and phospholipid metabolism of the yeast Saccharomyces cerevisiae. Biochim. Biophys. Acta 1801 (2010) 1314-1322

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Molecular Biology Group Group leader: Karin Athenstaedt Postdoctoral Fellow: Andreas Beranek Acyltransferases catalyzing triacylglycerol synthesis in the oleaginous yeast Yarrowia

lipolytica

The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to accumulate huge amounts of triacylglycerols (TAG). Along with other neutral lipids, TAG are stored in so-called lipid particles, cell compartments with a rather simple structure. A neutral lipid core mainly formed of TAG and steryl esters is surrounded by a phospholipid monolayer with few proteins embedded. By growing Yarrowia lipolytica cells in media containing, e.g., industrial fats or glycerol as a carbon source, the amount of TAG can be increased up to 40% of cell dry weight. This ability leads to its application in biotechnological processes such as single cell oil production or production of nutrients enriched in essential fatty acids which can serve as nutritional complements. However, Yarrowia lipolytica may also serve as a model organism to study lipid turnover in adipocytes, since not only the ability to store excessive amounts of TAG in lipid particles but also the composition of this cell compartment resembles adipocytes of higher eukaryotes. Despite these potentials of Yarrowia lipolytica information about TAG (lipid) metabolism in this yeast is rather limited. Thus, we started to investigate the proteome of Yarrowia lipolytica for proteins involved in TAG synthesis. Homology searches with TAG synthases of other eukaryotes as queries highlighted two candidate gene-products of the oleaginous yeast potentially catalyzing the formation of TAG. A decreased amount of TAG in mutant cells defective in these candidate genes already pinpointed to a function of these polypeptides in TAG formation. To investigate whether these candidate genes encode true TAG synthases these genes were heterologously expressed in cells of a Saccharomyces

cerevisiae mutant defective in neutral lipid synthesis and as a consequence lacking lipid particles (Fig. 1).

Figure 1: Restoration of lipid particle formation upon heterologous expression of Yarrowia

lipolytica TAG synthase candidates in mutant cells of the budding yeast Saccharomyces

cerevisiae lacking lipid particles. In contrast to the negative control (B; mutant + empty plasmid), lipid particles are formed in mutant cells transformed with plasmids bearing either of the respective candidate genes of Yarrowia lipolytica (C, D). Panel A shows lipid particle formation in a wild-type cell of Saccharomyces cerevisiae. Lipid particles are indicated by arrows. Size bar: 10 µm. Fluorescent microscopic inspection and lipid analyses of the respective mutants clearly demonstrated that these Yarrowia lipolytica genes encode true TAG synthases. The

B A C D

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characteristics of these two TAG synthases of the oleaginous yeast are currently under investigation. Biosynthesis of phosphatidic acid in yeast

Phosphatidic acid is of utmost importance, since it is the key intermediate for the formation of all glycerolipids, namely glycerophospholipids (membrane lipids) and triacylglycerols (storage lipids). Furthermore, this lipid functions in cell signaling. In all eukaryotic organisms enzymes catalyzing phosphatidic acid biosynthesis occur in redundancy. One reason for the existence of isoenzymes may be the formation of different phosphatidic acid pools supplying different pathways. In this project this hypothesis is tested in the model organism yeast Saccharomyces cerevisiae, which contains two glycerol-3-phosphate acyltransferases, Gat1p and Gat2p, and two 1-acyl glycerol-3-phosphate acyltransferases, Slc1p and Lpt1p. Glycerol-3-phosphate acyltransferases catalyze the first and rate limiting step in de novo synthesis of phosphatidic acid via the glycerol-3-phosphate pathway, namely the acylation of glycerol-3-phosphate yielding 1-acyl glycerol-3-phosphate (lyso-phosphatidic acid). A subsequent acylation converts lyso-phosphatidic acid to phosphatidic acid and is catalyzed by a 1-acyl glycerol-3-phosphate acyltransferase. The precise localization of glycerol-3-phosphate acyltransferases tagged with green fluorescent protein (GFP) has been determined by fluorescence microscopy and Western blot analyses with highly purified cell fractions of the respective mutants. Whereas Gat1p is associated with lipid particles and the endoplasmic reticulum, Gat2p is exclusively localized to the latter compartment in wild-type background (Fig. 2).

Most interestingly, the absence of either glycerol-3-phosphate acyltransferase results in a different distribution pattern of its counterpart, and in growth defects. Whether these phenotypes are caused by alterations in the lipid pattern of the respective mutants is currently under investigation. International cooperations

T. Chardot, Institut National de la Recherche Agronomique, Institut Jean-Pierre Bourgin,

UMR1318, Versailles, France J.-M. Nicaud, Institut National de la Recherche Agronomique, Laboratoire de Microbiologie

et Génétique Moléculaire, UMR1319, Jouy-en-Josas, France

B

Wild-type + GFP-Gat2p

Wild-type + GFP-Gat1p

A LP

LP

Figure 2: Fluorescence microscopy of Gat1p and Gat2p, respectively, tagged with green fluorescent protein (GFP) in wild-type background. A: GFP-Gat1p localizes to the endoplasmic reticulum and lipid particles (indicated by arrows). In contrast, GFP-Gat2p is only present in the endoplasmic reticulum (B).

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Research projects

FWF P21251: Biosynthesis of phosphatidic acid in yeast Publications

1) Athenstaedt, K.:

Neutral lipids in yeast: synthesis, storage and degradation. In Microbiology of Hydrocarbons, Oils, Lipids, and Derived Compounds. (Ed. by K. N. Timmis) Springer-Heidelberg, 2010, 471-480.

2) Athenstaedt, K.: Players in the neutral lipid game – proteins involved in neutral lipid metabolism in yeast. In Microbiology of Hydrocarbons, Oils, Lipids, and Derived Compounds. (Ed. by K. N. Timmis) Springer-Heidelberg, 2010, 537-546.

3) Athenstaedt, K.: Isolation and characterization of lipid particles of yeast. In Microbiology of Hydrocarbons, Oils, Lipids, and Derived Compounds. (Ed. by K. N. Timmis) Springer-Heidelberg, 2010, 4223-4229.

4) Athenstaedt, K.: Neutral lipid metabolism in yeast as a template for biomedical research. In Microbiology of Hydrocarbons, Oils, Lipids, and Derived Compounds. (Ed. by K. N. Timmis) Springer-Heidelberg, 2010, 3381-3382.

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Biophysical chemistry group

Group leader: Albin Hermetter Postdoctoral Fellow: Heidemarie Ehammer PhD students: Ute Stemmer, Daniel Koller, Claudia Ramprecht, Lingaraju Marlingapla Halasiddappa, Bojana Stojcic Master students: Andreas Gasser, Gabriel Pürstinger, Hannah Jaritz Technicians: Elfriede Zenzmaier

General description

Our research deals with the role of glycero(phospho)lipids and lipid modifying enzymes as components of membranes and lipoproteins, their function as mediators in cellular (patho)biochemistry, and their application as analytical tools in enzyme technology. Fluorescence spectroscopy is used as a main technique to investigate the behaviour of these biomolecules in the respective supramolecular systems. Section 1 of the following report summarizes our studies on lipid oxidation, the effects of oxidized lipids on intracellular signalling, and the inhibition of these processes by synthetic and natural antioxidants. Section 2 describes the development of fluorescence for functional proteomic analysis of lipolytic enzymes in microbial, animal and human cells.

1. Lipid oxidation and atherosclerosis – Lipotoxicity

1.1. Interaction of oxidized phospholipids with vascular cells

Sphingomyelin

Ceramide

acid

Sphingomyelinase

Oxidized Phospholipids

JNKp38 MAPKCaspase 3

O

O

O

O

O

O

OP

OH

O

ON

-+

O

O

O

O

O

H

OP

OH

O

N+

O

ERKAKT/PKB

NFkB

PROLIFERATIONSURVIVAL

OXPHOS-EuroMEMBRANE

Interactions of oxidized lipoproteins with the cells of the arterial wall induce and influence the progress of atherosclerosis. Accumulation of foam cells originating from macrophages and excessive intimal growth of vascular smooth muscle cells (SMC) alternating with focal massive cell

death are characteristics typical of the atherosclerotic lesion. These phenomena are largely mediated by the oxidized phospholipid components of the modified particles that are generated under the conditions of oxidative stress. In the framework of the research consortia LIPOTOX (SFB) and OXPHOS (EuroMEMBRANE), we investigate the “Toxicity of oxidized phospholipids in macrophages” and “The protein targets and apoptotic signaling of oxidized phospholipids”. These studies aim at identifying the molecular and cellular effects of an important subfamily of the oxidized phospholipids containing long hydrocarbon chains in

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position sn-1 and short polar acyl residues in position sn-2 of the glycerol backbone. The respective compounds trigger an intracellular signaling network including sphingomyelinases, (MAP) kinases and transcription factors thereby inducing proliferation or apoptosis of vascular cells. Both phenomena largely depend on the specific action of sphingolipid mediators that are acutely formed upon cell stimulation by the oxidized lipids. Fluorescence microscopic studies on labeled lipid analogs revealed that the short-chain phosphatidylcholines are easily transferred from the aqueous phase into the cell plasma membrane and eventually spread throughout the cells. Under these circumstances, the biologi-

cally active compounds are very likely to interfere directly with various signaling components inside the cells, which finally decide about cell growth or death. Investigations are being performed on the levels of the lipidome, the apparent enzyme activities, the proteome an the transcriptome to find the primary molecular targets and their downstream elements that are the key compnents of lipid-induced cell death.

1.2. Oxidative stress and antioxidants Antioxidants protect biomolecules against oxidative stress and thus, may prevent its pathological consequences. Specific fluorescent markers have been established for high-throughput screening of lipid and protein oxidation and its inhibition by natural and synthetic antioxidants. These methods can also be used for the determination of antioxidant capacities of biological fluids (e.g. serum) and edible oils. A prominent example is pumpkin seed oil which contains a variety of antioxidants (e.g. tocopherols and phenolic substances) and other secondary plant components that possess useful biological properties.

2. Functional proteomic analysis of lipolytic enzymes

The functional properties of lipases and phospholipases are the subject of our studies in the framework of the joint research program GOLD-Genomics of Lipid-associated Disorders at KFU Graz. Lipolytic enzymes catalyze intra- and extracellular lipid degradation. Dysfunctions in lipid metabolism may lead to various diseases including obesity, diabetes or atherosclerosis. In chemistry, lipases and esterases are important biocatalysts for (stereo)selective reactions on synthetic and natural substrates leading to defined products for pharmaceutical or agrochemical use.

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2.1. Fluorescent suicide inhibitors for in-gel analysis of lipases and phospholipases

Fluorescent suicide inhibitors have been developed as activity recognition probes (ARPs) for qualitative and quantitative analysis of active lipolytic enzymes in complex biological samples (industrial enzyme preparations, serum, cells, tissues). Since inhibitor binding to the active sites of lipases and esterases is specific and stoichiometric, accurate information can be obtained about the type of enzyme and the moles of active protein (active sites) in electrophoretically pure or heterogeneous enzyme preparations.

Active

Inactive

Inactive

Inactive

Inactive NBDRG

BG

NBDRG

BG

Inhibited

RG

BG

NBD

: Re a c t i v e p h os p h on a t e g r o up , inhibits the catalytic serine

: Binding group: is specifically recognized by the active enzyme

: fluorescent tag

irreversibly

488 nm; 540 nmλ λex em: :

Labelling of enzymes with an ARP specific for serine hydrolases

Fluorescent inhibitors are currently applied to proteomic analysis of the lipolytic enzymes in human and animal cells. These studies are performed in the framework of the joint project GOLD (Genomics of Lipid-associated Disorders/ coordinated by KFU Graz) which aims at discovering novel genes, processes and pathways that regulate lipid homeostasis in humans, mice and yeast. This is one of the projects in the field of functional genomics (GEN-AU, GENome research in AUstria) funded by the Austrian Federal Ministry for Education, Science and Culture (bm:bwk).

Green: wt Red: ko Red: wt Green: ko

DABGE Analysis of

lipases and esterases

in mouse adipose tissue

of wt and lipase ko

mice

Differential activity-based gel electrophoresis (DABGE) was developed for comparative analysis of two lipolytic proteomes in one polyacrylamide gel. For this purpose, the active lipases/esterases of two different samples are labelled with fluorescent inhibitors that possess identical substrate analogous structures but carry different cyanine dyes as reporter fluorophores. After sample mixing and protein separation by 1-D or 2-D PAGE, the enzymes carrying the sample-specific colors are detected and quantified. This technique can be used

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for the determination of differences in enzyme patterns, e.g. due to effects of genetic background, environment, metabolic state and disease.

Master Theses completed:

Andreas Gasser: Determination of substrate- and stereoselectivity of lipases The elucidation of the reaction mechanisms of lipolytic enzymes requires the determination of the substrate- and stereoselectivities of these proteins. In order to find a more straightforward alternative to the common assays using radioactive substrates, a method using chiral, pyrene-labelled substrates was developed in this study, which allows the determination of substrate- and stereoselectivity of lipases. This technique avoids the application of radioactive isotopes and as a consequence time consuming procedures. It is the basis for a simple and fast determination of lipase activity in high throughput analysis. On the basis of the robust and well characterized fungal Rhizomucor miehei lipase, the fluorescent substrates were tested for their applicability to determine substrate- and stereoselectivity of lipolytic enzymes using simple thin-layer chromatography. In order to obtain quantitative results and higher throughput, the method was adapted to HPLC analysis. This version was used to analyze activities as well as e.e. values of Rhizomucor miehei lipase, Chromobacterium viscosum lipase and Candida cylindracea cholesterol esterase. After overexpression in COS-7 cells, the selectivity and activity of the animal lipases adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL) and monoglyceride lipase (MGL) were measured in total cell lysates. Lipolytic activities were also determined in adipose tissue homogenates of ATGL- and HSL-deficient mice and compared to the wild type animals. The deficiency in the respective lipases correlated with the degradation patterns of the fluorescent substrates as determined by the above described method. Franziska Vogl: Transfection of macrophages with siRNA and plasmids. Expression and silencing of proteins targeted by oxidized phospholipids Oxidized phospholipids (oxPL) are generated from (poly)unsaturated glycerophospholipids in membranes and lipoproteins under conditions of oxidative stress. These compounds induce apoptosis in the cells of the vascular wall. In a previous functional proteomics study, the primary protein targets of a toxic aldehydophospholipid were identified in macrophages. To find out which of these protein candidates are involved in lipid-mediated cell death, we are following two different strategies. The first one uses siRNA technology to knock down the target proteins and measure the effect on cell death and signalling. The second approach is based on fusion proteins containing the protein candidates and RFP to measure Förster-resonance-energy-transfer (FRET) and determine the spatial proximity to fluorescently labelled oxPL. In this diploma thesis, protocols for chemical transfection of RAW 264.7 cells with expression plasmids were optimized. Five fluorescent fusion proteins were cloned and expressed in Cos-7 cells. Chemical transfection and electroporation were used to transfect cells with siRNA against GAPDH and Caspase-3. Transfections and silencing were monitored using FACS-measurements, Western blotting and qPCR and proved to be efficient in both cases. Silencing of caspase-3 was associated with a decrease in protein expression and loss of function. As a consequence, enzyme knock-down rendered the cells more resistant towards oxPL-induced apoptosis, supporting the assumption that the protease is causally involved in programmed cell death.

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International cooperations:

T. Hianik, Department of Biophysics and Chemical Physics, Comenius University, Bratislava,

Slovakia T. Hornemann, Institute for Clinical Chemistry, University Hospital Zürich; Zürich,

Switzerland M. Hof, Department of Biophysical Chemistry, Academy of Sciences of the Czech Republic,

Prague D. Russell, Department of Microbiology & Immunology, College of Veterinary Medicine;

Cornell University, Ithaca, USA I. Parmryd, Cell Biology, The Wenner-Gren Institute, Stockholm University, Stockholm,

Sweden T. Hugel, IMETUM, TU München, Garching, Germany P. Kinnunen, Biophysics, Aalto University, Helsinki, Finland Research projects:

BM.W_F Genomforschung in Österreich (GEN-AU): Exploring the lipolytic proteome.

Phospholipases (GOLD 3 project-Genomics of Lipid-associated Disorders, coordinated by KFU Graz).

FWF Special Research Program (SFB) LIPOTOX (coordinated by KFU Graz): Toxicity of oxidized phospholipids in macrophages.

FWF Doctoral Program Molecular Enzymology: Functional enzyme analysis. FWF EUROCORES-EuroMEMBRANE-OXPHOS-Protein targets and apoptotic signaling of

oxidized phospholipids Invited lectures: 1. Functional proteomic analysis of hydrolytic enzymes relevant to the stability and

activity of oxidized phospholipids in macrophages. Cellular radical stress and related biomarkers, COST Chemistry CM0603, Athens, Greece, February 22, 2010

2. Molecular and supramolecular targets of oxidized phospholipids. ESF Euromembrane Meeting, Heidelberg, Germany, April 7, 2010

3. Primary targets of oxidized phospholipids in macrophages. 5th Prague- Wroclaw Seminar on Biophysics, Prague, Czech Republic, November 18, 2010

Publications:

1. Landre JB, Blaess MF, Kohl M, Schlicksbier T, Ruryk A, Kinscherf R, Claus RA,

Hermetter A, Keller M, Bauer M, Deigner HP Addressable Bipartite Molecular Hook (ABMH): Immobilized Hairpin Probes with Sensitivity below 50 Femtomolar Anal.Biochem. 397, 60-66, 2010.

2. Sattler W, Nusshold C, Kollroser M, Köfeler H, rechberger G, reicher H, Üllen A, Bernhart E,Waltl S,Kratzer I,Hermetter A,Hackl H, Trajanoski Z,Hrzenjak A, Malle E Hypochlorite modification of sphingomyelin generates chlorinated lipid species that induce apoptosis and proteome alterations in dopaminergic PC12neurons in vitro Free Radical Biol Med 48, 1588-1600, 2010.

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3. Vanderven BC, Hermetter A, Huang A, Maxfield FR, Russell DG, Yates RM Development of a novel, cell-based chemical screen to identify inhibitors of intraphagosomal lipolysis in macrophages Cytometry A 77, 751-760, 2010.

4. Watschinger K, Keller MA, Golderer G, Hermann M, Maglione M, Sarg B, Lindner HH, Hermetter A, Werner-Felmayer G, konrat R, Hulo, N, Werner ER Identification of the gene encoding alkylglycerol monooxygenase defines a third class of tetrahydrobiopterin dependent enzymes PNAS, 107, 13672-13677, 2010.

5. Plochberger B, Stockner T, Chiantia S, Brameshuber M, Weghuber J, Hermetter A, Schwille P, Schuetz GJ Cholesterol slows down the lateral mobility of an oxidized phospholipid in a supported lipid bilayer Langmuir, 26, 17322-17329, 2010.

6. Schicher M, Morak M, Birner-Gruenberger R, Kayer H, Stojcic B, Rechberger G, Kollroser M, Hermetter A Functional proteomic analysis of lipases and esterases in cultured human adipocytes J. Proteome Res., 9, 6334-6344, 2010.

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Chemistry of Functional Foods Group leader: Michael Murkovic PhD students: Alam Zeb, Yuliana Reni Swasti Diploma students: Elham Fanaee Danesch, Tatjana Golubkova, Zivile Rocyte Technician: Alma Ljubijankic Honorary Professor: Klaus Günther, Forschungszentrum Jülich, Germany Visiting Professors: Evangelos Katzojannos, TEJ of Athens, Greece; Zhong Hayan, Central

South University of Forestry and Technology, China General description Antioxidants have different functions depending on the location of action. Is it the protection of biological systems maintaining the integrity of the system or the protection of foods against oxidation leading to health threatening substances? The exposure to oxidation products is either described as oxidative stress or the oxidized substances have an acute or chronic toxicity or are carcinogenic. The production of healthier and safer foods is of primary interest of this research group. The antioxidants of interest are polyphenols including anthocyanins and carotenoids. The evaluation of their occurrence in food and their behavior during processing and cooking is important especially when these substances are used as food additives. The safety evaluation of these compounds includes the evaluation of possible degradation products. Heating of food is a process that is normally done to improve the safety and digestibility and improve the sensory attributes like texture, color, and aroma. During the heating reactions occur that lead to the degradation of nutritive constituents like carbohydrates, proteins, amino acids and lipids. Some of the reaction products are contributing to the nice aroma, color, and texture of the prepared food and many of them are highly toxic and/or carcinogenic. However, these hazardous compounds occur in rather low concentrations being normally not acute toxic. The substances have a very diverse chemical background like heterocyclic amines, polycondensated aromatic compounds, acrylamide or furan derivatives. The aim of the research is to investigate the reaction mechanisms that lead to the formation of these hazardous compounds and establish strategies to mitigate the formation and thereby reducing the alimentary exposure. Carotenoids thermal oxidation in triacylglycerols

β-Carotene is one of most important and widespread carotenoids present in food. It is an important source of provitamin A. It acts as antioxidant in biological systems including the complex system of lipids. We studied the oxidation of β-carotene in model triacylglycerol systems, comparable to most of the high oleic edible oil. An HPLC electrospray ionization mass spectrometric method was developed for the separation and identification of triacylglycerols and its oxidation products (Figure 1). β-Carotene was found to act as antioxidant at lower temperature like 90°C and 100°C, while oxidation of triacylglycerols was closely correlated with the oxidation of β-carotene after 8 hours of thermal oxidation at 110 °C. The oxidized products of β-carotene were epoxides, peroxides and apocarotenals, separated and analyzed using C30 reversed phase HPLC-DAD and APCI-MS. We found that 110 °C and 120 °C were the favorable temperatures for studying the oxidation reaction of carotenoids, while 110 °C, 120 °C and 130 °C were favorable for triacylglycerol oxidation.

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Stability of triacylglycerols decreases with the increased formation of the oxidation products of β-carotene.

Time (min)

0 5 10 15 20 25 30

DAGs

HPLC-MS

200 300 400 500 600 700 800 900

Inte

nsity

0

20

40

60

80

100

879

901

896

599

288263

[ ]H+M +

[ ]4NH+M +

[ ]Na+M +

O

O

O

O

O

O

Trilinolein

LL+

m/z

200 300 400 500 600 700 800 900

Inte

nsity

0

20

40

60

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100903

898

599

288

263[ ]H+M +

[ ]4NH+M +

[ ]Na+M +

LL+

m/z

[ ]Li+H+O-M+

872

601

OL+

O

O

O

O

O

O

1-Oleoyl-2-linoleoyl-3-linoleoyl glycerol

881

200 300 400 500 600 700 800 900

Inte

nsity

0

20

40

60

80

100909

603

288 [ ]H+M+

[ ]4NH+M +

[ ]Na+M +

OO+

887

904

605

OS+

O

O

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O

1-Oleoyl-2-oleoyl-3-stearoyl glycerol

200 300 400 500 600 700 800 900

Inte

nsity

0

20

40

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603

263

[ ]H+M +

[ ]4NH+M +

[ ]Na+M +

OO+

m/z

[ ]Li+H+O-M +

876

883

Triolein

O

O

O

O

O

O

907

200 300 400 500 600 700 800 900

Inte

nsity

0

20

40

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100905

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603

288263

[ ]H+M +

[ ]4NH+M +

[ ]Na+M +

OO+

m/z

[ ]Li+H+O-M +

874

601

OL+

1-Oleoyl-2-linoleoyl-3-oleoyl glycerol

883

O

O

O

O

O

O

Figure 1. Separation and Identification of Triacylglycerols using HPLC-ESI-MS. Doctoral Thesis completed

Alam Zeb: Carotenoids and Triacylglycerols Interactions during Oxidation Carotenoids (β-carotene and astaxanthin) were oxidized in high oleic model triacylglycerols (TAGs) and edible oils such as corn and olive oils. The main techniques used in this dissertation were HPTLC and HPLC coupled to DAD and mass spectrometry. The previous literature on the uses of TLC suggests that HPTLC have the potential to be the first choice in the analysis of carotenoids in foods. We also found that HPTLC is a useful tool in the study of degradation of β-carotene in model TAGs and edible oils. The isocratic HPLC-ESI-MS method was very useful for the fast screening and identification of TAGs in edible oils. We correctly identify and separated thirteen, fourteen, fifteen and sixteen TAGs in refined olive oil, rapeseed oil, corn oil and sunflower oil, respectively. The oxidation products of TAGs were also studied using this method. Epoxy epidioxides, hydroxy bis-hydroperoxides and epidioxy bishydroperoxides were identified as major oxidized compounds that have been identified for the first time in model TAGs and edible oils under similar conditions. Other triacylglycerols oxidized species were hydroxy hydroperoxides, mono-hydroperoxides, bis-hydroperoxides, epoxy-epidioxides, and epoxides. Significant degradation of β-carotene was observed in sunflower oil. In high oleic model TAGs, β-carotene degraded significantly in the first three hours, however, in olive oil of relatively similar TAG composition, β-carotene degraded slowly. Astaxanthin degradation was much slower than β-carotene in olive oil. The HPLC method for the degradation and oxidation of carotenoids reveal a total of eight oxidized compounds of β-carotene in corn oil. The degradation of all-E-β-carotene in corn oil was relatively similar to model TAGs and olive oil. The interactions of carotenoids and TAGs reveal the pro-oxidant action of both β-carotene and astaxanthin. The pro-oxidant action of β-carotene was much stronger than astaxanthin. These findings help us to understand the

35

structural characterization of TAGs using mass spectrometry and the possible role and interactions of carotenoids or its oxidation products with the normal and oxidized triacylglycerols during thermal oxidation. Master Thesis completed

Elham Fanaee Danesch: Ethanol production from fruit waste with solid state fermentation using Saccharomyces cerevisiae

Disposal of agricultural wastes including rotten fruits and vegetables particularly in large cities and the lack of an appropriate method of recycling not only causes environmental pollution but also needs a loss of resources. These wastes are a good source of carbohydrates, acids, fibers, inorganic compounds and vitamins. Therefore they have a good potential for production of ethanol, animal feed, enzymes, pectin, etc. In this research, production of ethanol from fruit wastes by Saccharomyces cervisiae was investigated at 28 °C, pH 4.5 and 77 % moisture in solid state fermentation. S. cervisiae was obtained from Biochemical and Bioenvironmental Research Center (B.B.R.C.). A maximum ethanol production yield of 13.75 % based on the initial concentration was obtained from sugar after 22 h of fermentation which is equivalent to 1 g of ethanol per 42.35 g of fruit waste. The optimum value of effective parameters in production of ethanol were found to be 1 % ammonium sulfate, 1.5 % potassium dihydrogen phosphate, 2 % glucose and 41.06 g fruit waste. At the end of the fermentation process, 71 % of the substrate sugar was consumed. International cooperations

R. Venskutonis, Institute of Food Technology, Kaunas University of Technology, Lithuania T. Husoy, National Institute of Public Health, Olso, Norway H.R. Glatt, Deutsches Institute für Ernährungsforschung, Potsdam Rehbrücke, Germany E. Lazos, TEJ of Athens, Greece R. Grujic, University of East Sarajevo, Bosnia and Herzegovina E. Habul, University of Sarajevo, Bosnia and Herzegovina E. Winkelhausen, S. Kuzmanova, University Ss Cyril and Methodius, Skopie, FRYM H. Pinheiro, Instituto Superior Tecnico, Lisboa, Portugal V. Piironen, Department of Applied Chemistry and Microbiology, Helsinki, Finland M.J. Cantalejo, Department of Food Technol., Public University of Navarre, Pamplona, Spain Z. Cieserova, Food Research Institute, Bratislava, Slovakia C. Thongkraung, Prince of Songkla University, Hatyai, Thailand Research project

European Network of Excellence: EuroFIR European Food Information Resource Lectures

1) A. Zeb ß-Carotene induced oxidation of high oleic triacylglycerols model system. Österreichische Lebensmittelchemikertage. Schloss Seggau-Leibnitz, 19 May 2010

36

2) M. Murkovic Zusatzstoffe - unverzichtbare Begleiter in der Küche Wissenschaft mit Geschmack. Graz, 16 June 2010

3) A. Zeb Role of Astaxanthin in Thermal Oxidation of High Oleic Triacylglycerols. 8th Euro Fed Lipid Congress. Munich, 21 Nov. 2010

Publications

1) Zeb, A.; Murkovic, M.: Analysis of triacylglycerols in refined edible oils by isocratic HPLC-ESI-MS. European journal of lipid science and technology 112 (2010) 8, S. 844 – 851

2) Jöbstl, D.; Husoy, T.; Alexander, J.; Bjellaas, T.; Leitner, E.; Murkovic, M.: Analysis of 5-hydroxymethyl-2-furoic acid (HMFA) the main metabolite of alimentary 5-hydroxymethyl-2-furfural (HMF) with HPLC and GC in urine. Food Chemistry 123 (2010), S. 814 - 818

3) Prasetyo, E. N.; Kudanga, T.; Steiner, W.; Murkovic, M.; Wonisch, W.; Nyanhongo, G. S.; Gübitz, G.: Cellular and plasma antioxidant activity assay using tetramethoxy. Free radical biology & medicine 49 (2010), S. 1205 - 1211

4) Zeb, A.; Murkovic, M.: Characterization of the effects of β-Carotene on the thermal oxidation of Triacylglycerols using HPLC-ESI-MS. European journal of lipid science and technology 112 (2010) 11, S. 1218 - 1228

5) Zeb, A.; Murkovic, M.: High-Performance Thin-Layer Chromatographic Method for Monitoring the Thermal Degradation of β-Carotene in Sunflower Oil. Journal of Planar Chromatography - modern TLC 23 (2010) 1, S. 35 - 39

6) Nugroho Prasetyo, E.; Kudanga, T.; Steiner, W.; Murkovic, M.; Nyanhongo, G. S.; Gübitz, G.: Laccase-generated tetramethoxy azobismethylene quinone (TMAMQ) as a tool for antioxidant activity measurement. Food Chemistry 118 (2010) 2, S. 437 - 444

7) Zeb, A.; Murkovic, M.: Thin-Layer Chromatographic Analysis of Carotenoids in Plant and Animal Samples. Journal of Planar Chromatography - modern TLC 23 (2010) 2, S. 94 - 103

Scientific Conference

Biannual Conference of the Austrian Food Chemists Austrian Chemical Society – WG Food Chemistry, Cosmetics, and Tensides 19.5. – 21.5.2010, Schloss Seggau, Leibnitz, Austria

37

Lectures and Laboratory Courses

Aktuelle Trends und Ergebnisse in der Analytischen Chemie und Lebensmittelchemie

2 VO Günther K

Anleitung zu wissenschaftlichen Arbeiten aus Biochemie und Molekularer Biomedizin

2 SE Daum G, Hermetter A, Macheroux P

Anleitung zu wissenschaftlichen Arbeiten aus Biochemie und Molekularer Biomedizin

2 SE Daum G, Hermetter A, Macheroux P

Bioanalytik 2.25 VO Hermetter A, Klimant I Biochemie I 3.75 VO Macheroux P Biochemie II 1.5 VO Macheroux P Biophysikalische Chemie 1 2 PV Laggner P Biophysikalische Chemie 2 2 PV Laggner P Biophysikalische Chemie der Lipide 1 2 PV Hermetter A Biophysikalische Chemie der Lipide 2 2 PV Hermetter A Chemie und Technologie der Lebensmittel II 2 VO Murkovic M Chemische Veränderungen in Lebensmitteln I 2 PV Murkovic M Chemische Veränderungen in Lebensmittel II 2 PV Murkovic M Chemische Veränderungen in Lebensmitteln bei der Verarbeitung

2 SE Murkovic M

DissertantInnenseminar 1 1 SE Faculty DissertantInnenseminar 2 1 SE Faculty Enzymatische und mikrobielle Verfahren in der Lebensmittelherstellung

2 VO Murkovic M

Fisch- und Fischprodukte 1 VO Murkovic M Fluoreszenztechnologie 2 VO Hermetter A Fluoreszenztechnologie 1.5 LU Hermetter A GL Biochemie (BMT) 2 VO Macheroux P, Waldner-Scott I GL Chemie (BMT) 2 VO Hermetter A GL der Pharmakologie 2 VO Dittrich P Immunologische Methoden 2 VO Daum G Immunologische Methoden 2 LU Binter A, Daum G, Knaus T,

Wallner S Immunologische Methoden 1 VO Daum G Lebensmittelbiotechnologie 1.3 VO Murkovic M Lebensmittelchemie/Technologie 4 VU Leitner E, Murkovic M LU aus Biochemie I 5.33 LU Binter A, Daum G, Hermetter

A, Knaus T, Macheroux P, Waldner-Scott I, Wallner S

LU aus Biochemie II 4 LU Binter A, Daum G, Macheroux P, Waldner-Scott I, Wallner S

LU aus Molekularbiologie 3 LU Knaus T, Waldner-Scott I Membran Biophysik 1 2 PV Lohner K Membran Biophysik 2 2 PV Lohner K Membran-Mimetik 1 VO Lohner K Molekulare Enzymologie 2 VO Gruber K, Macheroux P,

Nidetzky B Molekulare Enzymologie I 2 PV Macheroux P Molekulare Enzymologie II 2 PV Macheroux P Molekulare Gastronomie und 1 EV Murkovic M

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Lebensmittelverarbeitung II Molekulare Physiologie 2 VO Macheroux P Physikalische Methoden in der Biochemie 2 LU Laggner P Physikalische Methoden in der Biochemie 1 VO Laggner P Projektlabor Bioanalytik 1 6 PR Daum G, Hermetter A,

Macheroux P Projektlabor Bioanalytik 2 6 PR Daum G, Hermetter A,

Macheroux P Projektlabor Biochemie und Molekulare Biomedizin 1

9 PR Trajanoski Z, Macheroux P, Daum G, Hermetter A,

Waldner-Scott I Projektlabor Biochemie und Molekulare Biomedizin 2

9 PR Daum G, Hermetter A, Macheroux P, Trajanoski Z,

Waldner-Scott I Projektlabor Lebensmittelbiotechnologie 1 6 PR Leitner E, Murkovic M Projektlabor Lebensmittelbiotechnologie 2 6 PR Leitner E, Murkovic M Seminar zu den LU aus Molekularbiologie 1 SE Athenstaedt K, Waldner-Scott I Spezielle Kapitel der Biochemie 1 VO Daum G, Hermetter A,

Macheroux P Spezielle Kapitel der Lebensmittelchem.- u. technologie I

2 SE Murkovic M

Spezielle Kapitel der Lebensmittelchemie- und techn.II

2 SE Murkovic M

Strukturanalyse in Biophysik u. Materialforschung

2 VO Laggner P

Unterrichtspraxis 2 SE Faculty Unterrichtspraxis 2 SE Faculty Wissenschaftliches Kolloquium für DissertantInnen 1

1 SE Faculty

Wissenschaftliches Kolloquium für DissertantInnen 2

1 SE Faculty

Zellbiologie 1.5 VO Daum G Zellbiologie 1 1 SE Daum G Zellbiologie 2 1 SE Daum G Zellbiologie der Lipide 1 2 PV Daum G Zellbiologie der Lipide 2 2 PV Daum G

39