Staff Members of the Institute of Biochemistry, TU Graz http://www ...
Staff Members of the Institute of Biochemistry, TU Graz http://www ...
Staff Members of the Institute of Biochemistry, TU Graz http://www ...
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<strong>Staff</strong> <strong>Members</strong> <strong>of</strong> <strong>the</strong> <strong>Institute</strong> <strong>of</strong> <strong>Biochemistry</strong>, <strong>TU</strong> <strong>Graz</strong><br />
<strong>http</strong>://<strong>www</strong>.biochemistry.tugraz.at/<br />
Pr<strong>of</strong>essors<br />
Peter Macheroux (Full Pr<strong>of</strong>essor & Head <strong>of</strong> <strong>the</strong> <strong>Institute</strong>)<br />
(peter.macheroux@tugraz.at; Tel.: +43-(0)316-873-6450)<br />
Gün<strong>the</strong>r Daum (Associate Pr<strong>of</strong>essor)<br />
(guen<strong>the</strong>r.daum@tugraz.at; Tel.: +43-(0)316-873-6462)<br />
Albin Hermetter (Associate Pr<strong>of</strong>essor)<br />
(albin.hermetter@tugraz.at; Tel.: +43-(0)316-873-6457)<br />
Assistants<br />
Dr. Karin A<strong>the</strong>nstaedt<br />
(karin.a<strong>the</strong>nstaedt@tugraz.at, Tel.:+43-(0)316-873-6460)<br />
Dr. Sigrid Deller<br />
(Sigrid.deller@tugraz.at; Tel.: +43-(0)316-873-6955)<br />
Mag. rer. nat. Gernot Rauch<br />
(gernot.rauch@tugraz.at; Tel.: +43-(0)316-873-6454)<br />
Mag. rer. nat. Heidemarie Ehammer<br />
(h.ehammer@tugraz.at, Tel.: +43-(0)316-873-6463)<br />
Office<br />
Annemarie Portschy<br />
portschy@tugraz.at; Tel.: +43-(0)316-873-6451; Fax: +43-(0)316-873-6952<br />
Technical <strong>Staff</strong><br />
Claudia Hrastnik; claudia.hrastnik@tugraz.at; Tel.: +43-(0)316-873-6460<br />
Rosemarie Trenker-El-Toukhy; r.trenker-el-toukhy@tugraz.at; Tel.: +43-(0)316-873-<br />
6464<br />
Anna Prem; anna.prem@tugraz.at; Tel.: 43-(0)316-873-6467<br />
Martin Puhl; martin.puhl@tugraz.at; Tel.: 43-(0)316-873 6453<br />
Elfriede Zenzmaier; elfriede.zenzmaier@tugraz.at; Tel.: +43-(0)316-873-6467<br />
Eva Pointner; eva.pointner@tugraz.at; Tel.: +43-(0)316-873-6452<br />
Leo H<strong>of</strong>er (Workshop); Tel.: +43-(0)316-873-8431 or 8433<br />
1
Brief History <strong>of</strong> <strong>the</strong> <strong>Institute</strong> <strong>of</strong> <strong>Biochemistry</strong><br />
The <strong>Institute</strong> <strong>of</strong> <strong>Biochemistry</strong> and Food Chemistry was born out <strong>of</strong> <strong>the</strong> division <strong>of</strong> <strong>the</strong><br />
<strong>Institute</strong> <strong>of</strong> Biochemical Technology, Food Chemistry and Microchemistry <strong>of</strong> <strong>the</strong> former<br />
School <strong>of</strong> Technology <strong>Graz</strong>. Toge<strong>the</strong>r with all <strong>the</strong> o<strong>the</strong>r Chemistry <strong>Institute</strong>s, it was<br />
located in <strong>the</strong> old Chemistry Building on Baron MANDELL's ground, corner<br />
Technikerstrasse-Mandellstrasse.<br />
1929 The <strong>Institute</strong> <strong>of</strong> Technical <strong>Biochemistry</strong> and Microbiology moved to <strong>the</strong> building<br />
<strong>of</strong> <strong>the</strong> Fürstlich-Dietrichstein-Stiftung, Schlögelgasse 9, in which all <strong>the</strong> Bio-<br />
Sciences were <strong>the</strong>n concentrated.<br />
1945 Georg GORBACH - initially in <strong>the</strong> rank <strong>of</strong> a docent and soon <strong>the</strong>reafter as a.o.<br />
Pr<strong>of</strong>essor - took over to lead <strong>the</strong> <strong>Institute</strong>. The <strong>Institute</strong> was renamed <strong>Institute</strong> <strong>of</strong><br />
Biochemical Technology and Food Chemistry.<br />
1948 G. GORBACH was nominated Full Pr<strong>of</strong>essor and Head <strong>of</strong> <strong>the</strong> <strong>Institute</strong>. In<br />
succession <strong>of</strong> <strong>the</strong> famous <strong>Graz</strong> School <strong>of</strong> Microchemistry founded by PREGL<br />
and EMICH, Pr<strong>of</strong>. GORBACH was one <strong>of</strong> <strong>the</strong> most prominent and active leaders<br />
in <strong>the</strong> fields <strong>of</strong> microchemistry, microbiology and nutritional sciences. After<br />
World War II, questions <strong>of</strong> water quality and waste water disposal became<br />
urgent; hence, <strong>the</strong> group <strong>of</strong> <strong>the</strong> future Pr<strong>of</strong>. K. S<strong>TU</strong>NDL, which at that time was<br />
part <strong>of</strong> <strong>the</strong> <strong>Institute</strong>, was gaining importance. In addition, a division to fight dryrot<br />
supervised by Dr. KUNZE and after his demise by H. SALOMON, was also<br />
affiliated with <strong>the</strong> <strong>Institute</strong>.<br />
1955 In honour <strong>of</strong> <strong>the</strong> founder <strong>of</strong> Microchemistry and former Pr<strong>of</strong>essor <strong>of</strong> <strong>the</strong><br />
Technical University <strong>Graz</strong>, <strong>the</strong> extended laboratory was called EMICH-<br />
Laboratories. At <strong>the</strong> same time, <strong>the</strong> <strong>Institute</strong> was renamed <strong>Institute</strong> <strong>of</strong><br />
Biochemical Technology, Food Chemistry and Microchemistry.<br />
Lectures and laboratory courses were held in <strong>Biochemistry</strong>, Biochemical Technology,<br />
Food Chemistry and Food Technology, Technical Microscopy and Microchemistry. In<br />
addition, <strong>the</strong> <strong>Institute</strong> covered Technical Microbiology toge<strong>the</strong>r with Biological and<br />
Bacteriological Analysis - with <strong>the</strong> exception <strong>of</strong> Pathogenic Microorganisms - and a<br />
lecture in Organic Raw Materials Sciences.<br />
1970 After <strong>the</strong> decease <strong>of</strong> Pr<strong>of</strong>. GORBACH, Pr<strong>of</strong>. GRUBITSCH was appointed Head<br />
<strong>of</strong> <strong>the</strong> <strong>Institute</strong>. Towards <strong>the</strong> end <strong>of</strong> <strong>the</strong> sixties, <strong>the</strong> division for water and waste<br />
water disposal headed by Pr<strong>of</strong>. S<strong>TU</strong>NDL was drawn out <strong>of</strong> <strong>the</strong> <strong>Institute</strong> and<br />
established as an <strong>Institute</strong> <strong>of</strong> its own. Pr<strong>of</strong>. SPITZY was nominated Pr<strong>of</strong>essor <strong>of</strong><br />
General Chemistry, Micro- and Radiochemistry. This division was also drawn<br />
out <strong>of</strong> <strong>the</strong> mo<strong>the</strong>r <strong>Institute</strong> and at <strong>the</strong> end <strong>of</strong> <strong>the</strong> sixties moved to a new building.<br />
1973 Division <strong>of</strong> <strong>the</strong> <strong>Institute</strong> for Biochemical Technology, Food Technology and<br />
Microchemistry took place. At first, Biochemical Technology toge<strong>the</strong>r with Food<br />
Technology formed a new <strong>Institute</strong> now called <strong>Institute</strong> <strong>of</strong> Biotechnology and<br />
Food Chemistry to which <strong>the</strong> newly nominated Pr<strong>of</strong>. LAFFERTY was appointed<br />
Head <strong>of</strong> <strong>the</strong> <strong>Institute</strong>.<br />
2
1973 Dr. F. PALTAUF, docent <strong>of</strong> <strong>the</strong> Karl-Franzens-University <strong>Graz</strong>, was nominated<br />
o. Pr<strong>of</strong>essor and Head <strong>of</strong> <strong>the</strong> newly established <strong>Institute</strong> <strong>of</strong> <strong>Biochemistry</strong>. The<br />
interest <strong>of</strong> Pr<strong>of</strong>. PALTAUF in studying biological membranes and lipids laid <strong>the</strong><br />
foundation for <strong>the</strong> future direction <strong>of</strong> research at <strong>the</strong> <strong>Institute</strong>. G. DAUM, S. D.<br />
KOHLWEIN, and A. HERMETTER joined <strong>the</strong> <strong>Institute</strong>. All three young<br />
scientists were given <strong>the</strong> chance to work as post docs in renowned laboratories in<br />
Switzerland and <strong>the</strong> USA: G. DAUM with <strong>the</strong> groups <strong>of</strong> G. Schatz (Basel,<br />
Switzerland) and R. Schekman (Berkeley, USA), A. HERMETTER with J. R.<br />
Lakowicz (Baltimore, USA) and S. D. KOHLWEIN with S. A. Henry (New<br />
York, USA). Consequently, independent research groups specialized in cell<br />
biology (G. D.), biophysics (A. H.) and molecular biology (S. D. K.) evolved at<br />
<strong>the</strong> <strong>Institute</strong> in <strong>Graz</strong>, with <strong>the</strong> group <strong>of</strong> Pr<strong>of</strong>. F. PALTAUF still focusing on <strong>the</strong><br />
chemistry and biochemistry <strong>of</strong> lipids.<br />
Teaching was always a major task <strong>of</strong> <strong>the</strong> <strong>Institute</strong>. Lectures, seminars and laboratory<br />
courses in basic biochemistry were complemented by special lectures, seminars, and<br />
courses held by <strong>the</strong> assistants who became docents in 1985 (G. D.), 1987 (A. H.), and<br />
1992 (S. D. K.). Lectures in food chemistry and food technology were held by C.<br />
WEBER and H. SALOMON, who were staff members <strong>of</strong> <strong>the</strong> <strong>Institute</strong>. Hence <strong>the</strong><br />
<strong>Institute</strong> was renamed <strong>Institute</strong> <strong>of</strong> <strong>Biochemistry</strong> and Food Chemistry.<br />
1990 The <strong>Institute</strong> moved to a new building at Petersgasse 12. Expansion <strong>of</strong> <strong>the</strong> size <strong>of</strong><br />
individual research groups and acquisition <strong>of</strong> new equipment were essential for<br />
<strong>the</strong> <strong>Institute</strong> to participate in novel collaborative efforts at <strong>the</strong> national and <strong>the</strong><br />
international level including joint projects (Forschungsschwerpunkte, Spezial-<br />
Forschungsbereiche) and EU-projects. Thus, <strong>the</strong> <strong>Institute</strong> <strong>of</strong> <strong>Biochemistry</strong>,<br />
toge<strong>the</strong>r with partner institutes from <strong>the</strong> Karl-Franzens-University was <strong>the</strong><br />
driving force to establish <strong>Graz</strong> as a centre <strong>of</strong> competence in lipid research.<br />
1993 W. PFANNHAUSER was appointed as Pr<strong>of</strong>essor <strong>of</strong> Food Chemistry. Through<br />
his own enthusiasm and engagement and that <strong>of</strong> his collaborators, this new<br />
section <strong>of</strong> <strong>the</strong> <strong>Institute</strong> rapidly developed and <strong>of</strong>fered students additional<br />
opportunities to receive a timely education.<br />
2000 The two sections, <strong>Biochemistry</strong> and Food Chemistry, being independent <strong>of</strong> each<br />
o<strong>the</strong>r with respect to personnel, teaching, and research, were separated. The new<br />
<strong>Institute</strong> <strong>of</strong> <strong>Biochemistry</strong> (Head Pr<strong>of</strong>. PALTAUF) and <strong>the</strong> new <strong>Institute</strong> <strong>of</strong> Food<br />
Chemistry and Food Technology (Head Pr<strong>of</strong>. PFANNHAUSER) evolved.<br />
2001 F. PALTAUF, who had been Full Pr<strong>of</strong>essor <strong>of</strong> <strong>Biochemistry</strong> and Head <strong>of</strong> <strong>the</strong><br />
Department for 28 years, retired in September 2001. G. DAUM was elected<br />
Head <strong>of</strong> <strong>the</strong> Department. S. D. KOHLWEIN was appointed Full Pr<strong>of</strong>essor <strong>of</strong><br />
<strong>Biochemistry</strong> at <strong>the</strong> Karl-Franzens University <strong>Graz</strong> and left <strong>the</strong> <strong>Institute</strong> <strong>of</strong><br />
<strong>Biochemistry</strong> <strong>of</strong> <strong>the</strong> <strong>TU</strong> <strong>Graz</strong>.<br />
2003 P. MACHEROUX was appointed Full Pr<strong>of</strong>essor <strong>of</strong> <strong>Biochemistry</strong> in September<br />
2003 and Head <strong>of</strong> <strong>the</strong> <strong>Institute</strong> <strong>of</strong> <strong>Biochemistry</strong> in January 2004. His research<br />
interests revolve around topics in <strong>the</strong> area <strong>of</strong> protein biochemistry and<br />
enzymology which shall streng<strong>the</strong>n <strong>the</strong> already existing activities at <strong>the</strong><br />
Technical University in this field (SFB Biocatalysis & Kplus).<br />
3
Highlights <strong>of</strong> 2007<br />
In April, Peter Macheroux’s group hosted a Cost B22 meeting on “Drug Target<br />
Characterisation” at Schloss Seggau, near <strong>Graz</strong>. This conference was dedicated to<br />
vitamin and c<strong>of</strong>actor biosyn<strong>the</strong>sis in protozoan parasites. The meeting was held from <strong>the</strong><br />
27 th to 29 th <strong>of</strong> April 2007 and was jointly organised by <strong>the</strong> members <strong>the</strong> EU-funded<br />
VITBIOMAL consortium. During <strong>the</strong> meeting, more than 30 experts from Europe,<br />
Australia and <strong>the</strong> United States discussed <strong>the</strong> potential to develop useful drugs against<br />
<strong>the</strong> biosyn<strong>the</strong>tic pathways <strong>of</strong> vitamins and drugs in protozoan parasites that cause severe<br />
health problems in tropical regions, such as malaria. In spring 2007, <strong>the</strong> FWF approved<br />
a project in P. Macheroux’s group to investigate enzymes involved <strong>of</strong> nikkomycin<br />
biosyn<strong>the</strong>sis. The aim <strong>of</strong> <strong>the</strong> three-year project is to unravel <strong>the</strong> enzymatic steps that<br />
lead from basic metabolites to aminohexuronic acid, which constitutes one <strong>of</strong> <strong>the</strong><br />
building blocks <strong>of</strong> peptidyl nucleosides such as nikkomycins produced by some<br />
Streptomycetes. The project is a joint effort between P. Macheroux’s group and Pr<strong>of</strong>.<br />
Gruber’s team at <strong>the</strong> <strong>Graz</strong> University and fur<strong>the</strong>r streng<strong>the</strong>ns <strong>the</strong> scientific interactions<br />
<strong>of</strong> NAWI <strong>Graz</strong> and <strong>the</strong> FWF-funded PhD program “Molecular Enzymology”.<br />
As in <strong>the</strong> years before, research in Gün<strong>the</strong>r Daum’s group was supported by several<br />
grants <strong>of</strong> <strong>the</strong> Austrian Science Fund (FWF). Teaching and administrative activities<br />
included appointment as a member <strong>of</strong> <strong>the</strong> Studienkommission Biomedical Engineering<br />
and chairman <strong>of</strong> <strong>the</strong> Doctoral School „Molecular <strong>Biochemistry</strong> and Biotechnology“ <strong>of</strong><br />
<strong>the</strong> <strong>TU</strong> <strong>Graz</strong>. As research related activities G. Daum worked as a Board Member <strong>of</strong> <strong>the</strong><br />
Austrian Science Fund (FWF), Associate Editor <strong>of</strong> FEMS Yeast Research, and Member<br />
<strong>of</strong> <strong>the</strong> Editorial Board <strong>of</strong> <strong>the</strong> Journal <strong>of</strong> Biological Chemistry. Moreover, he was elected<br />
Vice President <strong>of</strong> <strong>the</strong> Austrian Society <strong>of</strong> <strong>Biochemistry</strong> and Molecular Biology, and<br />
continued his activities as a Vice President <strong>of</strong> <strong>the</strong> International Conference on <strong>the</strong><br />
Bioscience <strong>of</strong> Lipids (ICBL) and Chairman <strong>of</strong> <strong>the</strong> Yeast Lipid Conference (YLC).<br />
Moreover, <strong>the</strong> European Federation for <strong>the</strong> Science and Technology <strong>of</strong> Lipids (Euro Fed<br />
Lipid) appointed G. Daum organizer <strong>of</strong> <strong>the</strong> Euro Fed Lipid Conference in <strong>Graz</strong>, Austria,<br />
in 2009. In 2007, invitations to distinguished research conferences in Italy, Finland and<br />
<strong>the</strong> USA were a great honour and an excellent opportunity to meet colleagues from <strong>the</strong><br />
same field <strong>of</strong> research and discuss problems <strong>of</strong> mutual interest.<br />
Albin Hermetter started a new project in <strong>the</strong> framework <strong>of</strong> <strong>the</strong> research consortium<br />
(SFB) LIPOTOX funded by <strong>the</strong> Austrian Science Fund (FWF). It is based on a<br />
functional genomic study and aims at identifying <strong>the</strong> molecular basis <strong>of</strong> “The toxicity <strong>of</strong><br />
oxidized phospholipids in macrophages” which is supposed to play a key role in <strong>the</strong><br />
development <strong>of</strong> a<strong>the</strong>rosclerosis. Since last year, A Hermetter is an Austrian<br />
representative in <strong>the</strong> COST action CM0603 which is devoted to “Bioradicals”. His<br />
contribution to <strong>the</strong> scientific program comprises various aspects <strong>of</strong> lipid oxidation and<br />
its biological consequences. The involvement <strong>of</strong> A. Hermetter’s group in industrial<br />
research and <strong>the</strong> continuous development <strong>of</strong> novel fluorescence technology have led to<br />
several patents during <strong>the</strong> last few years. Consequently, our institute became <strong>the</strong> fourth<br />
most successful department <strong>of</strong> <strong>TU</strong> <strong>Graz</strong> in <strong>the</strong> area <strong>of</strong> IPR and patenting.<br />
4
The PhD program “Molecular Enzymology” has received much attention recently as<br />
it has attempted to set new standards for <strong>the</strong> PhD education in <strong>Graz</strong> and beyond. Thanks<br />
to <strong>the</strong> generous financial endowment by <strong>the</strong> FWF, both universities, <strong>the</strong> state <strong>of</strong> Styria<br />
and <strong>the</strong> city <strong>of</strong> <strong>Graz</strong>, this “Doktoratskolleg” has been very successful. In January 2008,<br />
an application for an extension <strong>of</strong> <strong>the</strong> PhD program has been evaluated by an<br />
international board <strong>of</strong> reviewers. At present, seven PhD students <strong>of</strong> <strong>the</strong><br />
“Doktoratskolleg” are trained in our institute, all <strong>the</strong> more reason to keep our fingers<br />
crossed that an extension will be granted!<br />
5
<strong>Biochemistry</strong> group<br />
Group leader: Peter Macheroux<br />
Secretary: Annemarie Portschy<br />
Senior Research Scientist: Sigrid Deller<br />
PhD students: Asma Asghar (since December 1st), Alexandra Binter (since October<br />
1st), Heidi Ehammer, Karlheinz Flicker, Martina Neuwirth, Sonja Sollner, Gernot<br />
Rauch, Andreas Winkler<br />
Diploma students: Alexandra Binter (until September 30th), Katharina Fallmann, Daniel<br />
Koller, Sabrina Riedl, Markus Schober, Silvia Wallner<br />
Guest students: Sebastian H<strong>of</strong>zumahaus (FH Jülich, Germany), Nina Jajcanin (Ruder<br />
Boscovic <strong>Institute</strong>, Zagreb, Croatia),<br />
Sokrates student: Lucie Lorkova, <strong>Institute</strong> <strong>of</strong> Chemical Technology, Prague, Czech<br />
Republic<br />
Technicians: Rosemarie Trenker-El-Toukhy (karenziert), Eva Maria Pointner<br />
(karenziert), Anna Prem (Karenzvertretung), Martin Puhl (Karenzvertretung)<br />
General description<br />
The fundamental questions in <strong>the</strong> study <strong>of</strong> enzymes, <strong>the</strong> bio-catalysts <strong>of</strong> all living<br />
organisms, revolve around <strong>the</strong>ir ability to select a substrate (substrate specificity) and<br />
subject this substrate to a predetermined chemical reaction (reaction and regiospecificity).<br />
In general, only a few amino acid residues in <strong>the</strong> "active site" <strong>of</strong> an enzyme<br />
are involved in this process and hence provide <strong>the</strong> key to <strong>the</strong> processes taking place<br />
during enzyme catalysis. Therefore <strong>the</strong> focus <strong>of</strong> our research is to achieve a deeper<br />
understanding <strong>of</strong> <strong>the</strong> functional role <strong>of</strong> amino acids in <strong>the</strong> active site <strong>of</strong> enzymes with<br />
regard to substrate recognition and stereo- and regiospecificity <strong>of</strong> <strong>the</strong> chemical<br />
transformation. In addition, we are also interested in substrate-triggered conformational<br />
changes and how enzymes utilize c<strong>of</strong>actors (flavin, nicotinamide) to achieve catalysis.<br />
Towards <strong>the</strong>se aims we employ a multidisciplinary approach encompassing kinetic,<br />
<strong>the</strong>rmodynamic, spectroscopic and structural techniques. In addition, we use sitedirected<br />
mutagenesis to generate mutant enzymes to probe <strong>the</strong>ir functional role in <strong>the</strong><br />
before mentioned processes. Fur<strong>the</strong>rmore, we collaborate with our partners in academia<br />
and industry to develop inhibitors for enzymes, which can yield important new insights<br />
into enzyme mechanisms and can be useful as potential lead compounds in <strong>the</strong> design <strong>of</strong><br />
new drugs.<br />
The methods established in our laboratory comprise kinetic (stopped-flow and rapid<br />
quench analysis <strong>of</strong> enzymatic reactions), <strong>the</strong>rmodynamic (iso<strong>the</strong>rmal titration<br />
microcalorimetry) and spectroscopic (fluorescence, circular dichroism and UV/Vis<br />
absorbance) methods. We make also frequent use <strong>of</strong> MALDI-TOF and ESI mass<br />
spectrometry, protein purification techniques (chromatography and electrophoresis) and<br />
modern molecular biology methods to clone and express genes <strong>of</strong> interest to us. A brief<br />
description <strong>of</strong> our current research projects is given below.<br />
Berberine bridge enzyme<br />
Berberine bridge enzyme (BBE) is a central enzyme in <strong>the</strong> biosyn<strong>the</strong>sis <strong>of</strong> berberine, a<br />
pharmaceutically important alkaloid. The enzyme possesses a covalently attached FAD<br />
moiety, which is essential for catalysis. The reaction involves <strong>the</strong> oxidation <strong>of</strong> <strong>the</strong> N-<br />
6
methyl group <strong>of</strong> <strong>the</strong> substrate (S)-reticuline by <strong>the</strong> enzyme-bound flavin and<br />
concomitant formation <strong>of</strong> a carbon-carbon bond (<strong>the</strong> “bridge”). The ultimate acceptor <strong>of</strong><br />
<strong>the</strong> substrate-derived electrons is dioxygen, which reoxidizes <strong>the</strong> flavin to its resting<br />
state:<br />
H 3 C O<br />
HO<br />
H<br />
N<br />
CH 3<br />
OH<br />
OCH 3<br />
O 2 H 2 O 2<br />
berberine bridge enzyme<br />
(flavin-dependent)<br />
H 3 C O<br />
HO<br />
H<br />
N<br />
CH 2<br />
OH<br />
OCH 3<br />
(S)-reticuline<br />
(S)-scoulerine<br />
The BBE-catalysed oxidative carbon-carbon bond formation is a new example <strong>of</strong> <strong>the</strong><br />
versatility <strong>of</strong> <strong>the</strong> flavin c<strong>of</strong>actor in biochemical reactions. Our goal is to investigate this<br />
reaction in order to unravel its chemical mechanism and to elucidate <strong>the</strong> threedimensional<br />
structure <strong>of</strong> <strong>the</strong> protein. This will allow a detailed analysis <strong>of</strong> <strong>the</strong> structurefunction<br />
relationships in <strong>the</strong> enzyme active site and will enable us to probe <strong>the</strong><br />
mechanism by mutagenic analysis.<br />
Recently, we have developed a new expression system for BBE (using cDNA from<br />
Eschscholzia california, gold poppy) in Pichia pastoris, which produces fair amounts <strong>of</strong><br />
<strong>the</strong> protein. This work was accomplished in collaboration with Pr<strong>of</strong>. Toni Kutchan<br />
(Donald Danforth Plant Science Center, U. S. A.) and Pr<strong>of</strong>. Toni Glieder (<strong>TU</strong> <strong>Graz</strong>).<br />
The availability <strong>of</strong> suitable quantities <strong>of</strong> BBE enabled us to crystallise <strong>the</strong> protein and to<br />
solve <strong>the</strong> structure in collaboration with Pr<strong>of</strong>. Karl Gruber at <strong>the</strong> Karl-Franzens<br />
University <strong>Graz</strong> (see below).<br />
Based on <strong>the</strong> three-dimensional structure <strong>of</strong> BBE, we can now initiate a site-directed<br />
mutagenesis programme to investigate <strong>the</strong> role <strong>of</strong> amino acids present in <strong>the</strong> active site<br />
<strong>of</strong> <strong>the</strong> enzyme (<strong>the</strong>sis project <strong>of</strong> Andreas Winkler; diploma project <strong>of</strong> Sabrina Riedl).<br />
7
Chorismate synthase<br />
Chorismate synthase is <strong>the</strong> seventh enzyme <strong>of</strong> <strong>the</strong> shikimate pathway, which leads to<br />
aromatic amino acids and o<strong>the</strong>r aromatic metabolites. Chorismate synthase catalyses <strong>the</strong><br />
conversion <strong>of</strong> 5-enolpyruvylshikimate-3-phosphate to chorismate. The catalytic reaction<br />
requires reduced flavin for activity. This finding is very intriguing since flavins are<br />
essential c<strong>of</strong>actors in redox reactions and <strong>the</strong> elimination catalysed by chorismate<br />
synthase does not (formally) involve a reduction or oxidation <strong>of</strong> <strong>the</strong> substrate.<br />
Understanding <strong>the</strong> mechanism <strong>of</strong> chorismate synthase is <strong>of</strong> great importance because, as<br />
an enzyme <strong>of</strong> <strong>the</strong> shikimate pathway, it is only present in bacteria, fungi and plants<br />
which makes <strong>the</strong> enzyme an attractive target for <strong>the</strong> development <strong>of</strong> new antibiotics,<br />
fungicides and herbicides. More recently, <strong>the</strong> shikimate pathway was also discovered in<br />
apicomplexan parasites (Toxoplasma gondii, Plasmodium falciparum) emphasizing <strong>the</strong><br />
important role <strong>of</strong> this pathway as a potential drug target. Therefore a mechanistic and<br />
structural investigation <strong>of</strong> chorismate synthase may provide valuable information for <strong>the</strong><br />
development <strong>of</strong> compounds with antimicrobial, fungicidal, herbicidal or antimalarial<br />
activity. The mechanistic studies are currently concentrating on a site-directed<br />
mutagenesis program with <strong>the</strong> aim to understand <strong>the</strong> role <strong>of</strong> invariant amino acid<br />
residues in <strong>the</strong> active site <strong>of</strong> <strong>the</strong> enzyme. With this approach, we expect to gain fur<strong>the</strong>r<br />
insight into <strong>the</strong> functioning <strong>of</strong> <strong>the</strong> enzyme active site and <strong>the</strong> chemical reaction<br />
mechanism (<strong>the</strong>sis project <strong>of</strong> Gernot Rauch).<br />
Chorismate synthases can be classified according to <strong>the</strong> mode <strong>of</strong> reduction <strong>of</strong> <strong>the</strong><br />
required c<strong>of</strong>actor: mon<strong>of</strong>unctional chorismate synthase requires an external source <strong>of</strong><br />
reduced FMN whereas bifunctional enzymes possess an intrinsic NADPH:FMN<br />
oxidoreductase activity. The latter type <strong>of</strong> chorismate synthase has - so far - only been<br />
found in unicellular fungi (e.g. Saccharomyces cerevisiae and Neurospora crassa) and<br />
green algae (e.g. Euglena gracilis). Our current interest with regard to mono/bifunctionality<br />
<strong>of</strong> chorismate synthases focuses on <strong>the</strong> question whe<strong>the</strong>r mon<strong>of</strong>unctional<br />
chorismate synthases complement chorismate synthase deficient yeast (∆aro2). In order<br />
to tackle this issue, we have developed a system to evaluate complementation by<br />
mon<strong>of</strong>unctional bacterial and plant chorismate synthases in a ∆aro2 yeast strain. Our<br />
results indicate that bacterial and plant chorismate synthases are unable to confer<br />
prototrophy to <strong>the</strong> yeast knock-out despite expression <strong>of</strong> <strong>the</strong> proteins (see figure below,<br />
central panel; Ec, Escherichia coli; An, Anabaena; Le, Lycopersicon esculentum; Bs,<br />
Bacillus subtilis; Sc, Saccharomyces cerevisiae; Nc, Neurospora crassa; Tt,<br />
Tetrahymena <strong>the</strong>rmophila). This result suggests that <strong>the</strong> ability <strong>of</strong> chorismate synthase<br />
to utilize NADPH is essential for a fully functional shikimate pathway. This hypo<strong>the</strong>sis<br />
8
eceives fur<strong>the</strong>r support by additional experiments where co-complementation with a<br />
bacterial NADPH:FMN oxidoreductase (YcnD) led to restoration <strong>of</strong> prototrophy even in<br />
<strong>the</strong> case <strong>of</strong> mon<strong>of</strong>unctional chorismate synthases (see figure below, right panel) (<strong>the</strong>sis<br />
project <strong>of</strong> Heidi Ehammer).<br />
Nikkomycin biosyn<strong>the</strong>sis<br />
Nikkomycins are produced by several species <strong>of</strong> Streptomyces and exhibit fungicidal,<br />
insecticidal and acaricidal properties due to <strong>the</strong>ir strong inhibition <strong>of</strong> chitin synthase.<br />
Nikkomycins are promising compounds in <strong>the</strong> cure <strong>of</strong> <strong>the</strong> immunosuppressed, such as<br />
AIDS patients and organ transplant recipients as well as cancer patients undergoing<br />
chemo<strong>the</strong>rapy. Nikkomycin Z (R 1 = uracil & R 2 = OH, see below) is currently in clinical<br />
trial for its antifungal activity. Structurally, nikkomycins can be classified as peptidyl<br />
nucleosides containing two unusual amino acids, i.e. hydroxypyridylhomothreonine<br />
(HPHT) and aminohexuronic acid with an N-glycosidically linked base:<br />
Although <strong>the</strong> chemical structures <strong>of</strong> nikkomycins have been known since <strong>the</strong> 1970s,<br />
only a few biosyn<strong>the</strong>tic steps have been investigated in detail. The steps leading to <strong>the</strong><br />
aminohexuronic acid intermediate are unclear. Originally, it was hypo<strong>the</strong>sized that <strong>the</strong><br />
aminohexuronic acid moiety is generated by addition <strong>of</strong> an enolpyruvyl moiety from<br />
phosphoenolpyruvate (PEP) to ei<strong>the</strong>r <strong>the</strong> uridine or <strong>the</strong> 4-formyl-4-imidazolin-2-one<br />
analog to <strong>the</strong> 5’-position <strong>of</strong> ribose. This step is <strong>the</strong>n followed by ra<strong>the</strong>r speculative<br />
modifications to yield <strong>the</strong> aminohexuronic acid precursor. In contrast to this hypo<strong>the</strong>sis,<br />
we could recently demonstrate that UMP ra<strong>the</strong>r than uridine serves as <strong>the</strong> acceptor for<br />
<strong>the</strong> enolpyruvyl moiety, a reaction catalyzed by an enzyme encoded by a gene <strong>of</strong> <strong>the</strong><br />
nikkomycin operon termed nikO. Fur<strong>the</strong>rmore, we could demonstrate that it is attached<br />
to <strong>the</strong> 3’- ra<strong>the</strong>r than <strong>the</strong> 5’-position <strong>of</strong> UMP. These results are very intriguing since<br />
none <strong>of</strong> <strong>the</strong> nikkomycins syn<strong>the</strong>sized possess an enolpyruvyl group in this position <strong>of</strong><br />
<strong>the</strong> sugar moiety. Hence, it must be concluded that <strong>the</strong> resulting 3’-enolpyruvyl-UMP is<br />
subject to rearrangement reactions where <strong>the</strong> enolpyruvyl is detached from its 3’-<br />
position and transferred to <strong>the</strong> 5’-position <strong>of</strong> <strong>the</strong> ensuing aminohexuronic acid moiety.<br />
Currently, nothing is known about <strong>the</strong> enzymes involved in <strong>the</strong>se putative chemical<br />
steps. However, <strong>the</strong> genes that are co-transcribed with nikO have been reported and are<br />
designated nikI, nikJ, nikK, nikL, nikM and nikN. In order to investigate <strong>the</strong> role <strong>of</strong> <strong>the</strong><br />
9
encoded proteins in <strong>the</strong> biosyn<strong>the</strong>sis <strong>of</strong> <strong>the</strong> aminohexuronic acid moiety, <strong>the</strong>se genes<br />
will be cloned, expressed and <strong>the</strong> proteins purified for biochemical characterization<br />
(<strong>the</strong>sis project <strong>of</strong> Alexandra Binter and Asma Asghar; diploma project <strong>of</strong> Sebastian<br />
H<strong>of</strong>zumahaus).<br />
Proteins in a new vitamin B 6 biosyn<strong>the</strong>tic pathway<br />
Vitamin B 6 (pyridoxine) is an essential nutritional compound required by animals and<br />
humans, which lack <strong>the</strong> biosyn<strong>the</strong>tic pathway for its production. Only bacteria, fungi<br />
and plants possess <strong>the</strong> capability to syn<strong>the</strong>sise vitamin B 6 from basic suger components.<br />
The chemistry and biochemistry <strong>of</strong> <strong>the</strong> vitamin B 6 biosyn<strong>the</strong>sis has been studied in<br />
depth in <strong>the</strong> model bacterium Escherichia coli and it has been thought that this pathway<br />
is shared by all o<strong>the</strong>r organisms in which vitamin B 6 biosyn<strong>the</strong>sis occurs. In recent<br />
years, however, it emerged that <strong>the</strong> biosyn<strong>the</strong>tic pathway found in E. coli is limited to a<br />
small group <strong>of</strong> eubacteria (chiefly <strong>the</strong> γ-subdivision) and <strong>the</strong> majority <strong>of</strong> organisms have<br />
developed an entirely different route to generate vitamin B 6 .<br />
In our studies, we focus on two new proteins that are associated with <strong>the</strong> biosyn<strong>the</strong>sis <strong>of</strong><br />
vitamin B 6 in <strong>the</strong> prokaryote Bacillus subtilis: Pdx1 and Pdx2. The major goal <strong>of</strong> our<br />
studies is to unravel <strong>the</strong>ir interaction which results in <strong>the</strong> generation <strong>of</strong> pyridoxal<br />
phosphate. In order to achieve this, both proteins have been recombinantly produced and<br />
purified in quantities that afford <strong>the</strong> characterisation <strong>of</strong> binding by iso<strong>the</strong>rmal titration<br />
calorimetry.<br />
Moreover, we have cloned and expressed <strong>the</strong> Pdx1 homolog Snz1 from Saccharomyces<br />
cerevisiae. The purified protein was crystallized and <strong>the</strong> threedimensional structure<br />
solved by x-ray crystallography in collaboration with Dr. Ivo Tews, Biochemiezentrum<br />
Heidelberg. Interestingly, it was found that Snz1 exists as a hexamer ra<strong>the</strong>r than a<br />
dodecamer (see scheme below) and we are currently identifying <strong>the</strong> structural features<br />
that control oligomerization in <strong>the</strong> Pdx1 domain (<strong>the</strong>sis project <strong>of</strong> Martina Neuwirth and<br />
diploma project <strong>of</strong> Silvia Wallner).<br />
10
The absence <strong>of</strong> vitamin B 6 biosyn<strong>the</strong>sis in humans clearly suggests that <strong>the</strong> proteins<br />
involved are interesting new drug targets for <strong>the</strong> development <strong>of</strong> antibiotics, fungicides<br />
and herbicides. The recent discovery <strong>of</strong> vitamin B 6 biosyn<strong>the</strong>sis in <strong>the</strong> causative agent <strong>of</strong><br />
malaria, Plasmodium falciparum, prompted us to engage in an international<br />
collaboration in order to characterise <strong>the</strong> chemistry and enzymology <strong>of</strong> vitamin B 6<br />
biosyn<strong>the</strong>sis in this highly pathogenic organism. Our studies will provide a detailed<br />
understanding <strong>of</strong> <strong>the</strong> role(s) <strong>of</strong> Pdx1 and Pdx2 in P. falciparum and will enable us to<br />
initiate a drug design/screening programme which will receive additional support from<br />
<strong>the</strong> elucidation <strong>of</strong> <strong>the</strong> three-dimensional structures <strong>of</strong> <strong>the</strong> proteins and <strong>the</strong>ir active<br />
complex (<strong>the</strong>sis project <strong>of</strong> Karlheinz Flicker).<br />
NAD(P)H:FMN quinone reductases<br />
Following our mechanistic and structural studies on YcnD, a nitroreductase from<br />
Bacillus subtilis, we have cloned ano<strong>the</strong>r NADPH-dependent flavin containing<br />
oxidoreductase from this organism (termed YhdA). We have achieved high level<br />
expression in E. coli BL 21 host cells and purified it to homogeneity. Steady-state<br />
kinetic studies have shown that YhdA reduces FMN and nitro-organic compounds at <strong>the</strong><br />
expense <strong>of</strong> NADPH as <strong>the</strong> preferred electron donor. In parallel to <strong>the</strong> bacterial enzyme,<br />
we have also started to investigate <strong>the</strong> properties <strong>of</strong> a homologous enzyme from<br />
Saccharomyces cerevisiae (termed LOT6p). Despite <strong>the</strong> availability <strong>of</strong> a threedimensional<br />
x-ray structure for YhdA (1NNI) and LOT6p (1T0I), <strong>the</strong> physiological role<br />
<strong>of</strong> <strong>the</strong> enzyme was unclear. Our recent studies have now demonstrated that both<br />
enzymes rapidly reduce quinones at <strong>the</strong> expense <strong>of</strong> a reduced nicotinamide c<strong>of</strong>actor.<br />
These studies were complemented by localization studies <strong>of</strong> LOT6p in yeast, which<br />
confirmed <strong>the</strong> cytosolic occurrence <strong>of</strong> <strong>the</strong> protein (in collaboration with Pr<strong>of</strong>. Daum, <strong>TU</strong><br />
<strong>Graz</strong>). In order to fur<strong>the</strong>r characterize <strong>the</strong> cellular role <strong>of</strong> LOT6p, we are now in <strong>the</strong><br />
process to search for protein interaction partners (<strong>the</strong>sis project <strong>of</strong> Sonja Sollner and<br />
diploma project <strong>of</strong> Markus Schober).<br />
Interestingly, <strong>the</strong> bacterial homolog <strong>of</strong> LOT6p, YhdA, forms stable tetramers instead <strong>of</strong><br />
dimers. Apparently, this higher oligomeric state gives rise to increased <strong>the</strong>rmostability<br />
(Deller et al., 2006). Inspection <strong>of</strong> <strong>the</strong> three-dimensional structure led to <strong>the</strong><br />
identification <strong>of</strong> four salt-bridges, which hold two dimers toge<strong>the</strong>r (see figure).<br />
11
To investigate <strong>the</strong> importance <strong>of</strong> <strong>the</strong>se contacts for <strong>the</strong> formation <strong>of</strong> tetramers, we have<br />
generated a K109L and D137L single as well as a K109L/D137L double mutant protein.<br />
Currently, we are characterizing <strong>the</strong>se muteins with respect to oligomerisation,<br />
temperature stability and catalytic activity. With this approach we hope to contribute to<br />
<strong>the</strong> understanding <strong>of</strong> protein oligomerization in general (research project <strong>of</strong> Dr. Sigrid<br />
Deller and diploma project <strong>of</strong> Alexandra Binter).<br />
Diploma <strong>the</strong>sis completed<br />
Alexandra Binter: Characterization <strong>of</strong> Bacillus subtilis NADPH:FMN oxidoreductase<br />
YhdA in respect to oligomerization, activity and <strong>the</strong>rmostability.<br />
YhdA, a <strong>the</strong>rmostable NADPH:FMN oxidoreductase from Bacillus subtilis, shows<br />
quinone reductase activiy and occurs as a tetramer. The two extended dimer surfaces are<br />
packed against each o<strong>the</strong>r by a 90º rotation <strong>of</strong> one dimer with respect to <strong>the</strong> o<strong>the</strong>r. This<br />
assembly is stabilized by <strong>the</strong> formation <strong>of</strong> four salt bridges between K109 and D137. To<br />
investigate <strong>the</strong> importance <strong>of</strong> those ion pair contacts, <strong>the</strong> following muteins were created<br />
and heterologously expressed in E. coli: K109L and D137 single muteins and<br />
K109L/D137L and K109/D137K double muteins. These muteins were characterized<br />
with respect to oligomerization, showing that all <strong>the</strong> muteins form dimers in solution.<br />
Only K109L/D137L is partially tetrameric in concentrated solutions and tetrameric in<br />
crystals. Consequently, <strong>the</strong> salt bridges are essential for stabilizing <strong>the</strong> dimer-dimer<br />
interface. It was shown that <strong>the</strong>rmal denaturation <strong>of</strong> <strong>the</strong> dimeric muteins occurs at<br />
equally high temperatures as it does in <strong>the</strong> case <strong>of</strong> <strong>the</strong> wild-type protein. The activity as<br />
and NADPH:FMN oxidoreductase using oxygen and various quinones as substrates is<br />
reduced drastically compared to wild-type YhdA. Interestingly, <strong>the</strong> rate <strong>of</strong> <strong>the</strong> oxidative<br />
half reaction is similar to <strong>the</strong> wild-type, whereas <strong>the</strong> rate <strong>of</strong> <strong>the</strong> reductive half reaction is<br />
reduced significantly. Thus, it is concluded that binding <strong>of</strong> NADPH to <strong>the</strong> dimeric<br />
muteins is impaired.<br />
International cooperations<br />
M. Abramic, Ruder Boskovic <strong>Institute</strong> Zagreb, Croatia<br />
N. Amrhein and T. B. Fitzpatrick, <strong>Institute</strong> <strong>of</strong> Plant Sciences, ETH-Zürich, Switzerland<br />
S. Ghisla, Fachbereich Biologie, Universität Konstanz, Germany<br />
I. Jelesarov, Universität Zürich, Switzerland<br />
B. Kappes, Universitätklinikum Heidelberg, Germany<br />
T. Kutchan, Donald Danforth Plant Science Center, U.S.A.<br />
M. Groll, Technical University Munich, Germany<br />
B. A. Palfey, University <strong>of</strong> Michigan, U.S.A.<br />
I. Tews, Universität Heidelberg, Germany<br />
Research projects<br />
FWF P19858: “Enzyme der Nikkomycinbiosyn<strong>the</strong>sis”<br />
FWF P17215: “Proteins involved in a novel vitamin B 6 pathway”<br />
FWF P17471: “Mechanism <strong>of</strong> chorismate synthase”<br />
EU-STRP: “Vitamin biosyn<strong>the</strong>sis as target for antimalarial <strong>the</strong>rapy” (VitBioMal)<br />
12
FWF-Doktoratskolleg “Molecular Enzymology”<br />
WTZ Austria-Croatia: “Structure-function studies on metallopeptidases involved in<br />
metabolism <strong>of</strong> biologically active peptides”<br />
Invited lectures<br />
1) Cost B22 WG2 conference on “Drug target characterization” at Schloss Seggau,<br />
Austria (27.04.2007): “Thermodynamic protein-protein<br />
interaction studies on PfPdx1 and PfPdx2 from Plasmodium falciparum”.<br />
2) Universität Novi Sad, Serbia (04.06.2007): “Flavoenzyme catalysis in<br />
biosyn<strong>the</strong>tic reactions”.<br />
Publications<br />
1) Kommoju, P. R., Macheroux, P., and Ghisla, S.:<br />
Molecular cloning and expression <strong>of</strong> L-amino acid oxidase from <strong>the</strong> Malayan pit<br />
viper Calloselasma rhodostoma, Protein Expr Purific. 2007, 52:89-95.<br />
2) Sollner, S., Nebauer, R., Ehammer, H., Prem, A., Deller, S., Palfey, B. A.,<br />
Daum, G., and Macheroux, P.:<br />
LOT6 from Saccharomyces cerevisiae is a FMN-dependent reductase with a<br />
potential role in quinone detoxification, The FEBS Journal 2007, 274:1328-1339.<br />
3) Rauch, G., Ehammer, H., Bornemann, S., and Macheroux, P.:<br />
Mutagenic analysis <strong>of</strong> an invariant aspartate residue in chorismate synthase<br />
supports its role as an active site base, <strong>Biochemistry</strong> 2007, 46:3768-3774.<br />
4) Neuwirth, M., Flicker, K., Strohmeier, M., Tews, I., and Macheroux, P.:<br />
Thermodynamic characterization <strong>of</strong> <strong>the</strong> protein-protein interaction in <strong>the</strong><br />
heteromeric Bacillus subtilis pyridoxal synthase, <strong>Biochemistry</strong> 2007, 46:5131-<br />
5139.<br />
5) Hall, M., Stueckler, C., Kroutil, W., Macheroux, P., and Faber, K.:<br />
Asymmetric bioreduction <strong>of</strong> activated alkenes using cloned 12-oxophytodienoate<br />
reductase isoenzymes OPR-1 and OPR-3 from Lycopersicon esculentum<br />
(tomato): A striking switch <strong>of</strong> stereopreference, Angew. Chem. Int. Ed. 2007,<br />
46:3934-3937.<br />
6) Pricelius, S., Held, C., Sollner, S., Deller, S., Murkovic, M., Ullrich, R.,<br />
H<strong>of</strong>richter, M., Cavaco-Paulo, A., Macheroux, P., Guebitz, G. M.:<br />
Enzymatic reduction and oxidation <strong>of</strong> fibre-bound azo-compounds, Enzyme<br />
Microb. Techn. 2007, 40:1732-1738.<br />
7) Winkler, A., Kutchan, T. M., and Macheroux, P.:<br />
6-S-Cysteinylation <strong>of</strong> bi-covalently bound FAD in berberine bridge enzyme<br />
tunes <strong>the</strong> redox potential for optimal activity, J. Biol. Chem., 2007, 282:24437-<br />
24443.<br />
13
8) Ehammer, H., Rauch, G., Prem, A., Kappes, B., and Macheroux, P.:<br />
Conservation <strong>of</strong> NADPH utilization by chorismate synthase and its implications<br />
for <strong>the</strong> evolution <strong>of</strong> <strong>the</strong> shikimate pathway, Mol. Microbiol., 2007, 65:1249-<br />
1257.<br />
9) Fitzpatrick, T. B., Amrhein, A., Kappes, B., Macheroux, P., Tews, I., and<br />
Raschle, T.:<br />
Two independent routes <strong>of</strong> de novo vitamin B6 biosyn<strong>the</strong>sis: Not that different<br />
after all, Biochem. J., 2007, 407:1-13.<br />
10) Flicker, K., Neuwirth, M., Strohmeier, M., Kappes, B., Tews, I., and Macheroux,<br />
P.:<br />
Structural and <strong>the</strong>rmodynamic insights into <strong>the</strong> assembly <strong>of</strong> <strong>the</strong> heeromeric<br />
pyridoxalphosphate synthase from Plasmodium falciparum, J. Mol. Biol., 2007,<br />
374:732-748.<br />
11) Stueckler, C., Hall, M., Ehammer, H., Pointner, E., Kroutil, W., Macheroux, P.,<br />
and Faber, K.:<br />
Stereo-complementary bioreduction <strong>of</strong> α,β-unsaturated dicarboxylic acids and<br />
dimethly esters using enoate reductases: Enzyme- and substrate-based<br />
stereocontrol, Org, Lett., 2007, 9:5409-5411.<br />
Awards<br />
Gernot Rauch: Best paper award 2006/07 for doctoral students <strong>of</strong> <strong>the</strong> key research area<br />
“Life Science Technology” <strong>of</strong> <strong>the</strong> <strong>Graz</strong> University <strong>of</strong> Technolgy (July 2007)<br />
Sonja Sollner: Winner <strong>of</strong> <strong>the</strong> 1 st prize for best presentation at <strong>the</strong> annual graduate<br />
seminar <strong>of</strong> <strong>the</strong> PhD programme “Molecular Enzymology” (May 2007)<br />
14
Cell Biology Group<br />
Group leader: Gün<strong>the</strong>r Daum<br />
Graduate students: Sabine Rosenberger, Andrea Wagner, Tamara Wriessnegger,<br />
Irmgard Schuiki, Tibor Czabany, Melanie Connerth, Sona Rajakumari,<br />
Karlheinz Grillitsch, Miroslava Spanova<br />
Diploma student: Susanne Horvath<br />
Technician: Claudia Hrastnik<br />
Visiting scientists: Miroslava Spanova (Bratislava, Slovak Republic; Erasmus<br />
fellowship), Gordana Canadi Juresic (Rijeka, Croatia; CEEPUS fellowship)<br />
General description<br />
A fundamental problem <strong>of</strong> cell biology is <strong>the</strong> biogenesis <strong>of</strong> subcellular<br />
structures. Along that line, studies in our laboratory are focused on <strong>the</strong> syn<strong>the</strong>sis <strong>of</strong><br />
lipids and <strong>the</strong>ir assembly into organelle membranes <strong>of</strong> <strong>the</strong> yeast Saccharomyces<br />
cerevisiae. During <strong>the</strong> last decades, baker’s yeast has proven as a valuable and highly<br />
reliable model for eukaryotic cells. We make use <strong>of</strong> <strong>the</strong> advantages <strong>of</strong> this experimental<br />
system to combine biochemical, molecular and cell biological methods addressing<br />
problems <strong>of</strong> lipid metabolism and biomembrane formation.<br />
The majority <strong>of</strong> yeast lipids are syn<strong>the</strong>sized in <strong>the</strong> endoplasmic reticulum (ER)<br />
with some significant contributions <strong>of</strong> mitochondria, <strong>the</strong> Golgi and <strong>the</strong> so-called lipid<br />
particles. O<strong>the</strong>r subcellular fractions are devoid <strong>of</strong> lipid-syn<strong>the</strong>sizing activities. Spatial<br />
separation <strong>of</strong> lipid biosyn<strong>the</strong>tic steps and lack <strong>of</strong> lipid syn<strong>the</strong>sis in several cellular<br />
membranes necessitate efficient transfer <strong>of</strong> lipids from <strong>the</strong>ir site <strong>of</strong> syn<strong>the</strong>sis to <strong>the</strong>ir<br />
proper destination(s). The maintenance <strong>of</strong> organelle lipid pr<strong>of</strong>iles requires strict<br />
coordination <strong>of</strong> biosyn<strong>the</strong>tic and translocation activities. Specific aspects studied<br />
currently in our laboratory are (i) assembly and homeostasis <strong>of</strong><br />
phosphatidylethanolamine in yeast organelle membranes, (ii) neutral lipid storage in<br />
lipid particles and mobilization, and (iii) characterization <strong>of</strong> organelle membranes from<br />
Pichia pastoris.<br />
Phosphatidylethanolamine, a key component <strong>of</strong> yeast organelle membranes<br />
Work from our laboratory and from o<strong>the</strong>r groups had shown that phosphatidylethanolamine<br />
(PtdEtn), one <strong>of</strong> <strong>the</strong> major phospholipids <strong>of</strong> yeast membranes, is highly<br />
important for cellular function and cell proliferation. PtdEtn syn<strong>the</strong>sis in <strong>the</strong> yeast is<br />
accomplished by a network <strong>of</strong> reactions including (i) syn<strong>the</strong>sis <strong>of</strong> phosphatidylserine<br />
(PtdSer) in <strong>the</strong> endoplasmic reticulum, (ii) decarboxylation <strong>of</strong> PtdSer by mitochondrial<br />
phosphatidylserine decarboxylase 1 (Psd1p) or (iii) Psd2p in a Golgi/vacuolar<br />
compartment and (iv) <strong>the</strong> CDP-ethanolamine pathway (Kennedy pathway) in <strong>the</strong><br />
endoplasmic reticulum. To obtain more insight into biosyn<strong>the</strong>sis, assembly and<br />
homeostasis <strong>of</strong> PtdEtn single and multiple mutants bearing defects in <strong>the</strong> respective<br />
pathways can be used.<br />
Previous investigations in our laboratory were aimed at <strong>the</strong> molecular biological<br />
identification <strong>of</strong> novel components involved in PtdEtn homeostasis <strong>of</strong> <strong>the</strong> yeast<br />
Saccharomyces cerevisiae. For this purpose, a number <strong>of</strong> genetic screenings were<br />
15
performed. Three components identified through <strong>the</strong>se screenings were recently<br />
characterized in more detail, namely Oxa1p, Mrpl22p and Vps4p. Oxa1p is a<br />
component <strong>of</strong> <strong>the</strong> inner mitochondrial membrane and involved in <strong>the</strong> assembly <strong>of</strong><br />
proteins into this compartment (Figure 1). Deletion <strong>of</strong> OXA1 causes a defect in <strong>the</strong><br />
import <strong>of</strong> Psd1p, PtdSer decarboxylase 1, into mitochondria. Since Psd1p is <strong>the</strong> major<br />
enzyme <strong>of</strong> PtdEtn formation in <strong>the</strong> yeast, <strong>the</strong> oxa1 mutation results in a marked<br />
decrease <strong>of</strong> PtdEtn compared to wild type.<br />
Psd1p<br />
Targeting IM Sorting β-Subunit LGST -Subunit<br />
++++<br />
+<br />
TOM<br />
OMM<br />
TIM22<br />
TIM23<br />
Oxa1p<br />
Mba1p<br />
Psd1p<br />
IMM<br />
Psd1p<br />
Figure 1: The Oxa1p route <strong>of</strong> Psd1p import into mitochondria<br />
Mrpl22p is a component <strong>of</strong> mitochondrial ribosomes. Deletion <strong>of</strong> <strong>the</strong> respective<br />
gene causes defects comparable to psd1, namely decrease <strong>of</strong> cellular and mitochondrial<br />
PtdEtn and defects in mitochondrial respiration. The molecular reason for <strong>the</strong>se<br />
findings is not yet known. Vps4p is a component <strong>of</strong> <strong>the</strong> machinery governing protein<br />
traffic to yeast vacuoles. In strains deleted <strong>of</strong> VPS4 an alternative pathway <strong>of</strong> protein<br />
transport to vacuoles, <strong>the</strong> so-called autophagy/cytosol to vacuole targeting (Cvt)<br />
pathway, is induced. The latter route requires PtdEtn binding to Atg8p, a component<br />
involved in autophagy. Thus, shortage <strong>of</strong> PtdEtn in respective mutants compromises<br />
<strong>the</strong> autophagic process and causes a growth defect.<br />
To understand PtdEtn homeostasis in some more detail, we performed<br />
experiments defining traffic routes <strong>of</strong> PtdEtn within <strong>the</strong> yeast cell. For <strong>the</strong>se<br />
investigations we have chosen two subcellular fractions as destinations for PtdEtn<br />
translocation, namely peroxisomes and <strong>the</strong> plasma membrane. These two membranes<br />
have in common <strong>the</strong>ir lack <strong>of</strong> capacity to syn<strong>the</strong>size PtdEtn <strong>the</strong>mselves. We employed<br />
yeast mutants bearing defects in <strong>the</strong> three pathways <strong>of</strong> PtdEtn syn<strong>the</strong>sis, which are<br />
distributed among mitochondria, endoplasmic reticulum and Golgi. These experiments<br />
16
demonstrated that PtdEtn formed in all three organelles can be supplied to peroxisomes<br />
and to <strong>the</strong> plasma membrane. The fatty acid composition <strong>of</strong> peroxisomal and plasma<br />
membrane phospholipids was mostly influenced by <strong>the</strong> available pool <strong>of</strong> total species<br />
syn<strong>the</strong>sized, although a certain balancing effect was observed regarding <strong>the</strong> assembly <strong>of</strong><br />
PtdEtn species into <strong>the</strong> two target compartments. We assume that <strong>the</strong> phospholipid<br />
composition <strong>of</strong> peroxisomes and <strong>the</strong> plasma membrane is mainly affected by <strong>the</strong><br />
syn<strong>the</strong>sis <strong>of</strong> <strong>the</strong> respective components and subject to equilibrium, and to a lesser<br />
extent affected by specific transport and assembly processes. Organelle association and<br />
membrane contact between organelles may be a relevant mechanism for lipid transport<br />
between organelles, but components governing this process and related events have to<br />
be studied in future investigations.<br />
Neutral lipid storage in lipid particles and mobilization<br />
Yeast cells have <strong>the</strong> ability to store neutral lipids TAG (triacylglycerols) and SE<br />
(steryl esters) in subcellular structures named lipid particles/droplets. Upon requirement,<br />
TAG and SE can be mobilized and serve as building blocks for membrane biosyn<strong>the</strong>sis.<br />
In an ongoing project in our laboratory, we investigate and characterize enzymatic steps<br />
which lead to <strong>the</strong> storage <strong>of</strong> TAG and SE and at <strong>the</strong> same time to <strong>the</strong> biogenesis <strong>of</strong> lipid<br />
particles. Moreover, we study processes involved in <strong>the</strong> mobilization <strong>of</strong> TAG and SE<br />
depots.<br />
In <strong>the</strong> yeast, <strong>the</strong> SE synthases Are1p and Are2p, and <strong>the</strong> TAG synthases Dga1p<br />
and Lro1p contribute to <strong>the</strong> formation <strong>of</strong> lipid depots (Figure 2). We have to distinguish<br />
between de novo formation <strong>of</strong> lipid particles on one hand and incorporation <strong>of</strong> neutral<br />
lipids into preformed lipid particles on <strong>the</strong> o<strong>the</strong>r hand. Using a dga1 lro1 are1 are2<br />
quadruple mutant and <strong>the</strong> related set <strong>of</strong> triple mutants we performed cell biological and<br />
Figure 2: Pathways <strong>of</strong> triacylglycerol (TAG) and steryl ester (SE) metabolism in <strong>the</strong><br />
yeast. DAG: diacylglycerol; FFA: free fatty acids; PL: phospholipid.<br />
17
iochemical experiments to address questions related to new formation and growth <strong>of</strong><br />
lipid particles. Depending on <strong>the</strong> mutant used we were able to obtain lipid particles <strong>of</strong><br />
different composition. These different types <strong>of</strong> lipid particles were investigated using<br />
biochemical, cell biological and biophysical methods.<br />
Mutants described above were used to obtain detailed information about <strong>the</strong><br />
coordinate process <strong>of</strong> neutral lipid syn<strong>the</strong>sis and <strong>the</strong> specific contribution <strong>of</strong> each <strong>of</strong> <strong>the</strong><br />
four acyltransferases to lipid particle biogenesis. All four triple mutants with only one <strong>of</strong><br />
<strong>the</strong> gene products, Dga1p, Lro1p, Are1p or Are2p, active form lipid particles, although<br />
at different rate and lipid composition. Through <strong>the</strong>se experiments we showed that TAG<br />
or SE alone are sufficient for lipid particle biogenesis. Biophysical methods revealed<br />
that individual neutral lipids strongly affected <strong>the</strong> internal structure <strong>of</strong> lipid particles. SE<br />
form several ordered shells below <strong>the</strong> surface phospholipid monolayer <strong>of</strong> lipid particles,<br />
whereas TAG are more or less randomly packed in <strong>the</strong> center <strong>of</strong> <strong>the</strong> droplets (Figure 3).<br />
We propose that this structural arrangement <strong>of</strong> neutral lipids in lipid particles may be<br />
important for <strong>the</strong>ir physiological role especially with respect to mobilization <strong>of</strong> TAG<br />
and SE reserves.<br />
~5 shells SE TAG-core<br />
TG<br />
d~3500 Å<br />
d~ 200-<br />
300nm<br />
Figure 3: Internal structure <strong>of</strong> yeast lipid particles.<br />
A core <strong>of</strong> TAG is surrounded by several shells <strong>of</strong> SE. The surface <strong>of</strong> lipid particles<br />
consists <strong>of</strong> a phospholipid monolayer with proteins embedded.<br />
Similar to TAG and SE syn<strong>the</strong>sizing enzymes, proteins involved in degradation<br />
<strong>of</strong> neutral lipids occur in redundancy. Three TAG lipases named Tgl3p, Tgl4p and<br />
Tgl5p, and three SE hydrolases named Yeh1p, Yeh2p and Tgl1p were identified in <strong>the</strong><br />
yeast. Recently, we studied enzymatic properties <strong>of</strong> <strong>the</strong> three SE hydrolytic enzymes.<br />
Our investigations demonstrated that each SE hydrolase exhibits some substrate<br />
specificity. Using single, double and triple deletion mutants and strains overexpressing<br />
<strong>the</strong> three SE hydrolases, we showed <strong>the</strong> contribution <strong>of</strong> Tgl1p, Yeh1p and Yeh2p,<br />
respectively, to <strong>the</strong> mobilization <strong>of</strong> SE from <strong>the</strong>ir site <strong>of</strong> storage, <strong>the</strong> lipid particles. We<br />
also demonstrated that sterol intermediates stored as SE are readily mobilized through<br />
SE hydrolases, recycled to <strong>the</strong> sterol biosyn<strong>the</strong>tic pathway and converted to <strong>the</strong> final<br />
product, ergosterol. Finally, we propose that defects in SE cleavage affect sterol<br />
18
iosyn<strong>the</strong>sis in a type <strong>of</strong> feedback regulation. Conclusively, SE storage and mobilization<br />
although being dispensable for yeast viability under laboratory conditions contribute<br />
markedly to sterol homeostasis in this microorganism.<br />
Previous work from our laboratory had demonstrated that deletion <strong>of</strong> TGL3, <strong>the</strong><br />
major yeast TAG lipase located to lipid particles, resulted in a decreased mobilization <strong>of</strong><br />
triacylglycerols (TAG) from <strong>the</strong> lipid particles. TAG stored in a tgl3 mutant contained<br />
slightly increased amounts <strong>of</strong> C22:0 and C26:0 very long chain fatty acids (VLCFAs).<br />
These VLCFAs are indispensable for sphingolipid biosyn<strong>the</strong>sis and crucial for raft<br />
association in <strong>the</strong> yeast. To understand <strong>the</strong> possible role <strong>of</strong> TAG lipolysis in<br />
sphingolipid metabolism, we carried out radiolabeling experiments with sphingolipid<br />
precursors in wild type and tgl mutants. These results indicated that <strong>the</strong> mutants have a<br />
significantly reduced rate <strong>of</strong> sphingolipid biosyn<strong>the</strong>sis and an increased rate <strong>of</strong> turnover<br />
compared to wild type. Competition experiments with labeled palmitic acid (C16:0) and<br />
non-radiolabeled cerotic acid (C26:0) demonstrated that <strong>the</strong> C26:0 fatty acid competed<br />
with C16:0 fatty acid labeling <strong>of</strong> sphingolipids. Thus, we hypo<strong>the</strong>size that stored TAG<br />
can be a fatty acid donor for sphingolipid formation and TAG lipases contribute to this<br />
pathway <strong>of</strong> <strong>the</strong> yeast.<br />
Ano<strong>the</strong>r aspect addressed in our laboratory is characterization <strong>of</strong> yeast TAG<br />
lipases with special emphasis on <strong>the</strong> topology <strong>of</strong> <strong>the</strong>se proteins on <strong>the</strong> surface <strong>of</strong> lipid<br />
particles where <strong>the</strong>se proteins occur. This study is paralleled by topology studies <strong>of</strong><br />
o<strong>the</strong>r proteins located to <strong>the</strong> lipid particle surface, especially Erg1p, a squalene<br />
epoxidase, which has been studied in our laboratory before. Preliminary studies<br />
addressing accumulation <strong>of</strong> squalene in <strong>the</strong> yeast under certain conditions revealed that<br />
this component is localized to different subcellular sites depending on <strong>the</strong> cell biological<br />
and metabolic status <strong>of</strong> <strong>the</strong> yeast.<br />
Characterization <strong>of</strong> organelles from <strong>the</strong> yeast Pichia pastoris<br />
The yeast Pichia pastoris is an important experimental system for heterologous<br />
expression <strong>of</strong> proteins. Never<strong>the</strong>less, surprisingly little is known about organelles <strong>of</strong> this<br />
microorganism. For this reason, we started a systematic biochemical and cell biological<br />
study to establish standardized methods <strong>of</strong> Pichia pastoris organelle isolation and<br />
characterization. The expertise <strong>of</strong> our laboratory in cell fractionation and organelle<br />
isolation from <strong>the</strong> yeast Saccharomyces cerevisiae has been developed through a<br />
number <strong>of</strong> investigations in <strong>the</strong> past. Some <strong>of</strong> <strong>the</strong> methods applied for Saccharomyces<br />
cerevisiae can be used for Pichia pastoris cell fractionation, whereas o<strong>the</strong>rs cannot or<br />
need to be adapted.<br />
Recent work focused on <strong>the</strong> biochemical characterization <strong>of</strong> mitochondrial<br />
membranes from Pichia pastoris grown on different carbon sources (Figure 4). The<br />
major aims <strong>of</strong> this project were (1) qualitative and quantitative analysis <strong>of</strong><br />
phospholipids, fatty acids and sterols from mitochondrial membranes; and (2) analysis<br />
<strong>of</strong> mitochondrial subfractions from Pichia pastoris grown on different carbon sources.<br />
More recently, experiments were started to study selected lipid syn<strong>the</strong>sizing enzymes<br />
from Pichia pastoris with emphasis on <strong>the</strong> phosphatidylethanolamine biosyn<strong>the</strong>tic<br />
machinery. Preliminary data are a first step towards <strong>the</strong> understanding <strong>of</strong> <strong>the</strong> Pichia<br />
pastoris lipidome which will provide initial insight into <strong>the</strong> variety <strong>of</strong> membrane<br />
components <strong>of</strong> this yeast.<br />
19
A<br />
B<br />
V<br />
N<br />
M<br />
Figure 4: Electron microscopy <strong>of</strong> Pichia pastoris X33 wild type grown on glycerol (A).<br />
Under <strong>the</strong>se conditions mitochondria are well expressed. B: isolated mitochondria from<br />
Pichia pastoris grown on glycerol. M, mitochondria; V, vacuole; N, nucleus; Bars. 0.5<br />
and 2 µm. Pictures shown be curtsey <strong>of</strong> E. Ingolic, FELMI, <strong>TU</strong> <strong>Graz</strong>, Austria<br />
Doctoral Thesis completed<br />
Sabine Rosenberger<br />
Phosphatidylethanolamine (PtdEtn) is an essential component in <strong>the</strong> yeast<br />
Saccharomyces cerevisiae required for function and integrity <strong>of</strong> mitochondrial<br />
membranes, for protein import/assembly into <strong>the</strong> inner mitochondrial membrane, for<br />
GPI-anchor syn<strong>the</strong>sis and vacuolar targeting. Biosyn<strong>the</strong>sis <strong>of</strong> PtdEtn can be<br />
accomplished by three different pathways, with <strong>the</strong> major route mediated by<br />
phosphatidylserine decarboxylase 1 (Psd1p) residing in <strong>the</strong> inner mitochondrial<br />
membrane (IMM). Deletion <strong>of</strong> PSD1 leads to a significant decrease <strong>of</strong> cellular and<br />
mitochondrial PtdEtn <strong>the</strong>reby conferring a petite phenotype, which is characterized by<br />
<strong>the</strong> loss <strong>of</strong> respiratory capacity and mitochondrial DNA. To identify a link between <strong>the</strong><br />
petite phenotype and disturbed lipid homeostasis in mitochondria a petite yeast strain<br />
collection was screened for mutants with abnormal phospholipid patterns. This<br />
approach unveiled some candidate genes, among <strong>the</strong>m MRPL22. Deletion <strong>of</strong> MRPL22<br />
leads to a decreased amount <strong>of</strong> cellular and mitochondrial PtdEtn similar to a psd1<br />
deletion strain. Ano<strong>the</strong>r major part <strong>of</strong> this <strong>the</strong>sis was focused on <strong>the</strong> assembly <strong>of</strong> PtdEtn<br />
into peroxisomal membranes. To define <strong>the</strong> traffic routes <strong>of</strong> PtdEtn from <strong>the</strong>ir site <strong>of</strong><br />
syn<strong>the</strong>sis to peroxisomes single and multiple mutants bearing defects in <strong>the</strong> different<br />
pathways <strong>of</strong> PtdEtn syn<strong>the</strong>sis were used. PtdEtn formed through all three pathways in<br />
different subcellular membranes can be supplied to peroxisomes with comparable<br />
efficiency.<br />
International Cooperations<br />
J. Cregg, Keck Graduate <strong>Institute</strong> <strong>of</strong> Applied Life Sciences, Claremont, CA, USA<br />
I. Hapala, Slovak Academy <strong>of</strong> Sciences, <strong>Institute</strong> <strong>of</strong> Animal <strong>Biochemistry</strong> and Genetics,<br />
Ivanka pri Dunaji, Slovak Republic<br />
V. Tomekova, Department <strong>of</strong> Medical Chemistry, <strong>Biochemistry</strong> and Clinical<br />
<strong>Biochemistry</strong>, Faculty <strong>of</strong> Medicine UPJS, Košice, Slovak Republic<br />
20
T. Hoefken, <strong>Institute</strong> <strong>of</strong> <strong>Biochemistry</strong>, University <strong>of</strong> Kiel, Germany<br />
D. Warnecke, <strong>Institute</strong> <strong>of</strong> Botany, University <strong>of</strong> Hamburg, Germany<br />
I. Feussner, Plant <strong>Biochemistry</strong>, Albrecht-von-Haller-<strong>Institute</strong> <strong>of</strong> Plant Sciences, Georg-<br />
August University Göttingen, Germany<br />
R. Erdmann, <strong>Institute</strong> <strong>of</strong> Physiological Chemistry, Ruhr-University Bochum, Germany<br />
Research Projects<br />
FWF 17321: Phosphatidylethanolamine <strong>of</strong> yeast mitochondria<br />
FWF 18857: Neutral lipid storage and mobilization in <strong>the</strong> yeast Saccharomyces<br />
cerevisiae<br />
FWF L-178 (Translational research): Characterization <strong>of</strong> organelles from <strong>the</strong> yeast<br />
Pichia pastoris<br />
FWF PhD Program: Molecular Enzymology<br />
Invited Lectures<br />
1) G. Daum<br />
Phosphatidylethanolamine, a key lipid <strong>of</strong> <strong>the</strong> yeast<br />
Comenius University, Bratislava, Slovak Republic, 19 th April 2007<br />
2) G. Daum<br />
Neutral lipid storage and mobilization in <strong>the</strong> yeast<br />
Technical University, Bratislava, Slovak Republic, 20 th April 2007<br />
3) G. Daum, T. Czabany, A. Wagner, M. Spanova, S. Rajakumari, K. Grillitsch and<br />
K. A<strong>the</strong>nstaedt<br />
Playing <strong>the</strong> yeast lipid game<br />
8 th Yeast Lipid Conference, Torino, Italy, 10-12 May 2007<br />
4) G. Daum, K. A<strong>the</strong>nstaedt, A. Wagner, T. Czabany, K. Grillitsch, S. Rajakumari,<br />
M. Spanova, D. Zweytick and E. Ingolic<br />
Formation and mobilization <strong>of</strong> neutral lipid depots in <strong>the</strong> yeast<br />
3 rd European Federation <strong>of</strong> Biotechnology Conference on Physiology <strong>of</strong> Yeast and<br />
Filamentous Fungi, Helsinki, Finland, 13-16 June 2007<br />
5) G. Daum<br />
The Life Cycle <strong>of</strong> Neutral Lipids: Syn<strong>the</strong>sis, Storage and Degradation<br />
Gordon Research Conference “Molecular & Cellular Biology <strong>of</strong> Lipids“,<br />
Waterville Valley Resort, NH, USA, 22-27 July<br />
Publications<br />
1) Daum, G., Wagner, A., Czabany, T. and A<strong>the</strong>nstaedt, K.<br />
Dynamics <strong>of</strong> neutral lipid storage and mobilization in <strong>the</strong> yeast<br />
Biochimie 89 (2007) 243-248<br />
21
2) Sollner, S.; Nebauer, R.; Ehammer, H.; Prem, A.; Deller, S.; Palfey, B. A.; Daum,<br />
G. and Macheroux, P.<br />
Lot6p from Saccharomyces cerevisiae is a FMN-dependent reductase with a<br />
potential role in quinone detoxification<br />
FEBS J. 274 (2007) 1328-1339<br />
3) Czabany, T., A<strong>the</strong>nstaedt, K. and Daum, G.<br />
Syn<strong>the</strong>sis, storage and degradation <strong>of</strong> neutral lipids in yeast<br />
Biochim. Biophys. Acta 1771 (2007) 299-309<br />
4) Wriessnegger, T, Gübitz, G, Leitner, E, Ingolic, E, Cregg, J, de la Cruz, B. J. and<br />
Daum, G.<br />
Lipid composition <strong>of</strong> peroxisomes from <strong>the</strong> yeast Pichia pastoris grown on<br />
different carbon sources<br />
Biochim. Biophys. Acta 1771 (2007) 455-461<br />
5) Perktold, A., Zechmann, B., Daum, G. and Zellnig, G.<br />
Organelle association visualized by three-dimensional ultrastructural imaging <strong>of</strong><br />
<strong>the</strong> yeast cell<br />
FEMS Yeast Research 7(2007) 629-638<br />
6) Nebauer, R., Rosenberger, S. and Daum, G.<br />
Phosphatidylethanolamine, a limiting factor <strong>of</strong> autophagy in yeast strains bearing a<br />
defect in <strong>the</strong> carboxypeptidase y pathway <strong>of</strong> vacuolar targeting<br />
J. Biol. Chem. 282 (2007) 16736-16743<br />
7) Nebauer, R, Schuiki, I., Kulterer, B., Trajanoski, Z. and Daum, G.<br />
The phosphatidylethanolamine level <strong>of</strong> yeast mitochondria is affected by <strong>the</strong><br />
mitochondrial components Oxa1p and Yme1p<br />
FEBS J. 274 (2007) 6180-6190<br />
8) Daum, G, Wagner, A., Czabany, T, Grillitsch, K. and A<strong>the</strong>nstaedt, K.<br />
Lipid storage and mobilization pathways in yeast<br />
Novartis Foundation Symposium No.286 (2007), 142-154<br />
Award<br />
Ruth Nebauer, a former PhD student, received <strong>the</strong> Best Paper Award 2006/07 for<br />
doctoral students <strong>of</strong> <strong>the</strong> Key Research Area “Life Science Technology” <strong>of</strong> <strong>the</strong> <strong>Graz</strong><br />
University <strong>of</strong> Technology (July 2007)<br />
22
Molecular Biology Group<br />
Karin A<strong>the</strong>nstaedt<br />
Lipid particles – more than simple storage compartments<br />
(Pr<strong>of</strong>essorial dissertation)<br />
Lipid particles are intracellular compartments consisting <strong>of</strong> a neutral lipid core<br />
surrounded by a phospholipid monolayer with few proteins embedded. In former days,<br />
<strong>the</strong> only function ascribed to <strong>the</strong>se particles was that <strong>of</strong> a simple storage compartment<br />
containing backup molecules, mainly triacylglycerols and steryl esters, which are<br />
mobilized when nutrients are no longer provided by <strong>the</strong> environment. This view,<br />
however, had to be revised, since investigations during <strong>the</strong> last decade demonstrated<br />
active participation <strong>of</strong> lipid particle proteins in lipid metabolism. My work deals with<br />
identification and characterization <strong>of</strong> several lipid particle proteins <strong>of</strong> <strong>the</strong> yeast<br />
Saccharomyces cerevisiae which are involved in processes such as phosphatidic acid<br />
biosyn<strong>the</strong>sis, triacylglycerol and sterol formation, and triacylglycerol mobilization.<br />
Analysis <strong>of</strong> <strong>the</strong> lipid particle proteome <strong>of</strong> <strong>the</strong> oleaginous yeast Yarrowia lipolytica<br />
revealed that most proteins are counterparts <strong>of</strong> lipid particle polypeptides <strong>of</strong><br />
Saccharomyces cerevisiae. Comparison <strong>of</strong> <strong>the</strong> yeasts’ lipid particle proteome with that<br />
<strong>of</strong> higher eukaryotic cells indicates that participation in lipid metabolism is a general<br />
feature <strong>of</strong> this compartment.<br />
Is Nte1p <strong>of</strong> Saccharomyces cerevisiae a true member <strong>of</strong> <strong>the</strong> NTE1-family<br />
Neuropathy target esterase (NTE) was originally identified as <strong>the</strong> target <strong>of</strong><br />
organophosphorous esters (OP) present in many pesticides as well as warfare agents.<br />
Toxifications with OPs are causing delayed neuropathy in man and some o<strong>the</strong>r<br />
vertebrates. The syndrome <strong>of</strong> <strong>the</strong> organophosphorous-induced delayed neuropathy<br />
(OPIDN) is characterised by paralysis <strong>of</strong> <strong>the</strong> lower limbs and degeneration <strong>of</strong> long axons<br />
in <strong>the</strong> spinal cord. These effects are common responses <strong>of</strong> neurons in metabolic<br />
disorders (e.g., diabetes), toxic states and advanced age. Nei<strong>the</strong>r NTE nor its homologs<br />
in o<strong>the</strong>r organisms are closely related to any o<strong>the</strong>r esterase or protease. The importance<br />
<strong>of</strong> NTE can be seen by <strong>the</strong> fact that mice lacking this protein die as early embryos, and<br />
Figure 1: “Holes” in <strong>the</strong> brain <strong>of</strong> a sws-mutant <strong>of</strong> <strong>the</strong> fruit fly Drosophila melanogaster.<br />
First “holes” in <strong>the</strong> brain <strong>of</strong> a sws-mutant can be already observed after 5 days (B). The<br />
number <strong>of</strong> “holes” increases progressively with age (20d; C). In contrast, no “holes” can<br />
be observed in wild-type brains after 20 days (A).<br />
23
inactivation <strong>of</strong> SWS (swiss cheese), <strong>the</strong> homolog <strong>of</strong> NTE in <strong>the</strong> fruit fly Drosophila<br />
melanogaster, leads to a progressive degeneration <strong>of</strong> <strong>the</strong> nervous system, which can be<br />
observed as big “holes” in <strong>the</strong> brain <strong>of</strong> <strong>the</strong> fly.<br />
Heterologous expression <strong>of</strong> <strong>the</strong> murine NTE can completely substitute SWS in<br />
Drosophila, demonstrating <strong>the</strong> functional conservation <strong>of</strong> this protein. A homolog <strong>of</strong><br />
NTE is also present in yeast Saccharomyces cerevisiae encoded by <strong>the</strong> NTE1. In<br />
contrast to higher eukaryotes, deletion <strong>of</strong> this protein in <strong>the</strong> model organism yeast does<br />
not result in a phenotype under several standard conditions tested. However, due to <strong>the</strong><br />
presence <strong>of</strong> this polypeptide in a single cell organism, NTE must be involved in a more<br />
general context as in development, neural survival and brain integrity. All efforts to<br />
identify <strong>the</strong> biological function, substrate, and interaction partners <strong>of</strong> NTE in higher<br />
eukaryotes have failed so far. In contrast, Nte1p <strong>of</strong> <strong>the</strong> yeast has been reported to exhibit<br />
phospholipase B activity, thus indicating a similar function <strong>of</strong> <strong>the</strong> respective<br />
counterparts <strong>of</strong> higher eukaryotes. In this project, we are investigating whe<strong>the</strong>r Nte1p <strong>of</strong><br />
Saccharomyces cerevisiae is a true member <strong>of</strong> <strong>the</strong> NTE protein family and thus suitable<br />
as a model for unravelling <strong>the</strong> biological function <strong>of</strong> this protein family. In vitro, <strong>the</strong><br />
functional conservation between NTE <strong>of</strong> higher eukaryotes and Nte1p <strong>of</strong> <strong>the</strong> yeast has<br />
already been demonstrated. But is this also <strong>the</strong> case in vivo For answering this<br />
question, I expressed NTE1 <strong>of</strong> <strong>the</strong> yeast heterologously in <strong>the</strong> fruit fly Drosophila<br />
melanogaster. A functional compensations would be indicated by reversion <strong>of</strong> <strong>the</strong><br />
phenotype <strong>of</strong> an sws-mutant fly, i.e., observable as a decrease in <strong>the</strong> size and / or number<br />
<strong>of</strong> “holes” in <strong>the</strong> brain <strong>of</strong> Drosophila. Several different mutant lines were constructed<br />
expressing <strong>the</strong> yeast NTE1 to different extent and in different tissues. Subsequently, <strong>the</strong><br />
brains <strong>of</strong> <strong>the</strong>se mutants were analyzed by fluorescence microscopy for <strong>the</strong> presence <strong>of</strong><br />
“holes”. Any result from full reversion <strong>of</strong> <strong>the</strong> phenotype to no effect has been<br />
considered, however, <strong>the</strong> phenotype was even worse in <strong>the</strong> transfectants showing a<br />
higher number <strong>of</strong> “holes”. In addition, <strong>the</strong> life span <strong>of</strong> <strong>the</strong> sws-mutants expressing NTE1<br />
<strong>of</strong> yeast was significantly reduced compared to control flies. This result indicates that<br />
<strong>the</strong>re is no functional conservation among <strong>the</strong> NTE protein family in vivo. However, my<br />
searches for an explanation what caused this ra<strong>the</strong>r striking effect provided some<br />
evidence that Nte1p interacts with enzymes <strong>of</strong> <strong>the</strong> lipid metabolism and forms a protein<br />
complex with at least one more polypeptide. Preliminary results indicated that this is<br />
also true for Sws <strong>of</strong> <strong>the</strong> fruit fly. Fur<strong>the</strong>rmore, mutations in certain lipid syn<strong>the</strong>sizing<br />
pathways led to smaller and a fewer number <strong>of</strong> holes in <strong>the</strong> brain <strong>of</strong> a sws-mutant fly,<br />
thus indicating that <strong>the</strong> phenotype <strong>of</strong> <strong>the</strong> sws-mutant is caused by major changes in <strong>the</strong><br />
lipid pattern. Analysis <strong>of</strong> <strong>the</strong> lipid pattern <strong>of</strong> <strong>the</strong> respective mutant flies will reveal<br />
which lipid parameters are required for normal development <strong>of</strong> <strong>the</strong> nervous system, and<br />
will provide valuable information for potential targets to compensate effects caused by<br />
neurons in metabolic disorders (e.g., diabetes), toxic states and advanced age.<br />
This work was performed at OHSU (Oregon Health and Sciences University,<br />
Portland, Oregon) during my post doctoral studies in 2007.<br />
Lipid metabolism <strong>of</strong> <strong>the</strong> yeast Yarrowia lipolytica<br />
The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to produce<br />
and store triacylglycerols. For this reason, this microorganism has become attractive for<br />
industrial applications such as production <strong>of</strong> single cell oil. In a former project, we<br />
investigated lipid particles <strong>of</strong> <strong>the</strong> oleaginous yeast with respect to <strong>the</strong>ir lipid and protein<br />
composition. Analysis <strong>of</strong> <strong>the</strong> lipid particle proteome identified major lipid particle<br />
24
proteins. The “standard” proteome <strong>of</strong> lipid particles isolated from glucose grown cells<br />
was complemented by several o<strong>the</strong>rs when cells were grown on oleic acid. Homology<br />
searches using <strong>the</strong> ORFs coding for lipid particle polypeptides <strong>of</strong> Yarrowia lipolytica as<br />
a query revealed that most lipid particle proteins are counterparts <strong>of</strong> proteins involved in<br />
lipid metabolism in Saccharomyces cerevisiae. However, since <strong>the</strong> function <strong>of</strong> <strong>the</strong>se<br />
proteins was only deduced upon homology, we are currently investigating <strong>the</strong> “true”<br />
function <strong>of</strong> <strong>the</strong>se proteins with a special emphasis on those involved in triacylglycerol<br />
syn<strong>the</strong>sis.<br />
Figure 2:<br />
Accumulation <strong>of</strong> neutral<br />
lipids in lipid particles <strong>of</strong><br />
Yarrowia lipolytica wildtype<br />
cell ei<strong>the</strong>r grown on<br />
oleic acid (left) or glucose<br />
(right). Neutral lipids are<br />
stained with <strong>the</strong> fluorescent<br />
lipophilic dye NileRed®.<br />
Special Activities<br />
Habilitation for <strong>the</strong> subject <strong>Biochemistry</strong><br />
Postdoctoral studies at <strong>the</strong> Oregon Health and Sciences University (OHSU), Portland,<br />
Oregon, USA<br />
International Cooperations<br />
T. Chardot and J.-M. Nicaud, <strong>Institute</strong> National de la Recherche Agronomique, UMR<br />
Chimie Biologique, Thiverval Grignon, France<br />
D. Kretzschmar, Oregon Health and Sciences University, Portland, Oregon, USA<br />
J. M. Thevelein, Centre for Food and Microbial Technology, Department <strong>of</strong> Microbial<br />
and Molecular Systems, Katholeke Universiteit Leuven, Heverlee, Belgium<br />
Publications<br />
1) Daum, G., Wagner, A., Czabany, T. and A<strong>the</strong>nstaedt, K.<br />
Dynamics <strong>of</strong> neutral lipid storage and mobilization in <strong>the</strong> yeast<br />
Biochimie 89 (2007) 243-248<br />
2) Czabany, T., A<strong>the</strong>nstaedt, K. and Daum, G.<br />
Syn<strong>the</strong>sis, storage and degradation <strong>of</strong> neutral lipids in yeast<br />
Biochim. Biophys. Acta 1771 (2007) 299-309<br />
3) Daum, G, Wagner, A., Czabany, T, Grillitsch, K. and A<strong>the</strong>nstaedt, K.<br />
Lipid storage and mobilization pathways in yeast<br />
Novartis Foundation Symposium No.286 (2007), 142-154<br />
25
Biophysical chemistry group<br />
Group leader: Albin Hermetter<br />
Post-docs: Heidrun Susani-Etzerodt, Jo-Anne Rasmussen<br />
Graduate students: Maria Morak, Jessica Schatte, Maximilian Schicher, Ute Stemmer<br />
Undergraduate students: Peter Krempl, Andreas Gasser, Martina Geier<br />
Technicians: Elfriede Zenzmaier<br />
General description<br />
Our research deals with <strong>the</strong> role <strong>of</strong> glycero(phospho)lipids and lipid modifying enzymes<br />
as components <strong>of</strong> membranes and lipoproteins, as mediators in cellular<br />
(patho)biochemistry, and <strong>the</strong>ir application as analytical tools in enzyme technology.<br />
Fluorescence spectroscopy is used as a main technique to investigate <strong>the</strong> behaviour <strong>of</strong><br />
<strong>the</strong>se biomolecules in <strong>the</strong> respective supramolecular systems.<br />
Section 1 <strong>of</strong> <strong>the</strong> following report summarizes our studies on lipid oxidation, <strong>the</strong> effects<br />
<strong>of</strong> oxidized lipids on intracellular signalling, and <strong>the</strong> inhibition <strong>of</strong> <strong>the</strong>se processes by<br />
syn<strong>the</strong>tic and natural antioxidants.<br />
Section 2 describes <strong>the</strong> development <strong>of</strong> fluorescence and chip technology for functional<br />
proteomic analysis <strong>of</strong> lipolytic enzymes in microbial, animal and human cells.<br />
1. Lipid oxidation and a<strong>the</strong>rosclerosis – Lipotoxicity<br />
1.1. Interaction <strong>of</strong> oxidized phospholipids with vascular cells<br />
Sphingomyelin<br />
acid<br />
Sphingomyelinase<br />
Oxidized -<br />
Phospholipids<br />
O<br />
H<br />
O<br />
O<br />
O<br />
O<br />
O<br />
O<br />
O<br />
O<br />
O<br />
O P O<br />
OH<br />
O<br />
O<br />
O<br />
O P O<br />
OH<br />
+<br />
N<br />
N +<br />
Ceramide<br />
JNK<br />
p38 MAPK<br />
Caspase 3<br />
ERK<br />
AKT/PKB<br />
NFkB<br />
PROLIFERATION<br />
SURVIVAL<br />
Interactions <strong>of</strong> oxidized lipoproteins with<br />
<strong>the</strong> cells <strong>of</strong> <strong>the</strong> arterial wall induce and<br />
influence <strong>the</strong> progress <strong>of</strong> a<strong>the</strong>rosclerosis.<br />
Accumulation <strong>of</strong> foam cells originating<br />
from macrophages and excessive intimal<br />
growth <strong>of</strong> vascular smooth muscle cells<br />
(SMC) alternating with focal massive cell<br />
death are characteristics typical <strong>of</strong> <strong>the</strong> a<strong>the</strong>rosclerotic lesion. These phenomena are<br />
largely mediated by <strong>the</strong> oxidized phospholipid components <strong>of</strong> <strong>the</strong> modified particles that<br />
are generated under <strong>the</strong> conditions <strong>of</strong> oxidative stress. In <strong>the</strong> framework <strong>of</strong> <strong>the</strong> research<br />
consortium LIPOTOX, we investigate <strong>the</strong> “Toxicity <strong>of</strong> oxidized phospholipids in<br />
macrophages”. This study aims at identifying <strong>the</strong> molecular and cellular effects <strong>of</strong> an<br />
important subfamily <strong>of</strong> <strong>the</strong> oxidized phospholipids containing long hydrocarbon chains<br />
in position sn-1 and short polar acyl residues in position sn-2 <strong>of</strong> <strong>the</strong> glycerol backbone.<br />
The respective compounds trigger an intracellular signaling network including<br />
sphingomyelinases, (MAP) kinases and transcription factors <strong>the</strong>reby inducing<br />
proliferation or apoptosis <strong>of</strong> vascular cells. Both phenomena largely depend on <strong>the</strong><br />
26
specific action <strong>of</strong> sphingolipid mediators that are acutely formed upon cell stimulation<br />
by <strong>the</strong> oxidized lipids. Fluorescence microscopic studies on labeled lipid analogs<br />
revealed that <strong>the</strong> short-chain phosphatidylcholines are easily transferred from <strong>the</strong><br />
aqueous phase into <strong>the</strong> cell plasma membrane and eventually spread throughout <strong>the</strong><br />
cells.<br />
Under <strong>the</strong>se circumstances, <strong>the</strong> biologically<br />
active compounds are very likely to interfere<br />
directly with various signaling components<br />
inside <strong>the</strong> cells, which finally decide about cell<br />
growth or death. Investigations will be<br />
performed on <strong>the</strong> levels <strong>of</strong> <strong>the</strong> lipidome, <strong>the</strong><br />
apparent enzyme activities, <strong>the</strong> proteome an <strong>the</strong><br />
transcriptome to find <strong>the</strong> primary molecular<br />
targets and <strong>the</strong>ir downstream elements that are<br />
<strong>the</strong> key compnents <strong>of</strong> lipid-induced cell death.<br />
1.2. Oxidative stress and antioxidants<br />
Antioxidants protect biomolecules against<br />
oxidative stress and thus, may prevent its<br />
pathological consequences. Specific<br />
fluorescent markers have been established for<br />
high-throughput screening <strong>of</strong> lipid and protein<br />
oxidation and its inhibition by natural and<br />
syn<strong>the</strong>tic antioxidants. These methods can<br />
also be used for <strong>the</strong> determination <strong>of</strong><br />
antioxidant capacities <strong>of</strong> biological fluids (e.g.<br />
serum) and edible oils. A prominent example<br />
is pumpkin seed oil which contains a variety<br />
<strong>of</strong> antioxidants (e.g. tocopherols and phenolic<br />
substances) and o<strong>the</strong>r secondary plant<br />
components that possess useful biological<br />
properties.<br />
2. Functional proteomic analysis <strong>of</strong> lipolytic enzymes<br />
The functional properties <strong>of</strong> lipases and esterases are <strong>the</strong> subject <strong>of</strong> our studies in <strong>the</strong><br />
framework <strong>of</strong> two joint research programs (GOLD-Genomics <strong>of</strong> Lipid-associated<br />
Disorders at KFU <strong>Graz</strong> and <strong>the</strong> Research center Applied Biocatalysis at <strong>TU</strong> <strong>Graz</strong>).<br />
Lipolytic enzymes catalyze intra- and extracellular lipid degradation. Dysfunctions in<br />
lipid metabolism can lead to serious diseases including obesity, diabetes or<br />
a<strong>the</strong>rosclerosis. In chemistry, lipases and esterases are important biocatalysts for<br />
(stereo)selective reactions on syn<strong>the</strong>tic and natural substrates leading to defined<br />
products for pharmaceutical or agrochemical use.<br />
27
2.1. Fluorescent suicide inhibitors for in-gel analysis <strong>of</strong> lipases and esterases<br />
Fluorescent suicide inhibitors have been developed as activity recognition probes<br />
(ARPs) for qualitative and quantitative analysis <strong>of</strong> active lipolytic enzymes in complex<br />
biological samples (industrial enzyme preparations, serum, cells, tissues). Since<br />
inhibitor binding to <strong>the</strong> active sites <strong>of</strong> lipases and esterases is specific and<br />
stoichiometric, accurate information can be obtained about <strong>the</strong> type <strong>of</strong> enzyme and <strong>the</strong><br />
moles <strong>of</strong> active protein (active sites) in electrophoretically pure or heterogeneous<br />
enzyme preparations.<br />
Labelling <strong>of</strong> enzymes with an ARP specific for serine hydrolases<br />
BG<br />
Inactive<br />
Inactive<br />
RG NBD<br />
RG: Reacti v e p h osphonate gr o up,<br />
irreversibly inhibits <strong>the</strong> catalytic serine<br />
Active<br />
BG<br />
BG: Binding group: is specifically recognized<br />
by <strong>the</strong> active enzyme<br />
Inactive<br />
Inactive<br />
Inhibited<br />
RG<br />
NBD<br />
NBD : fluorescent tag<br />
λ : 488 nm; λ : 540 nm<br />
ex<br />
em<br />
Fluorescent inhibitors are currently applied to <strong>the</strong> proteomic analysis <strong>of</strong> <strong>the</strong> lipolytic<br />
enzymes in human and animal cells. These studies are performed in <strong>the</strong> framework <strong>of</strong><br />
<strong>the</strong> joint project GOLD (Genomics <strong>of</strong> Lipid-associated Disorders-coordinated by KFU<br />
<strong>Graz</strong>) which aims at discovering novel genes, processes and pathways that regulate lipid<br />
homeostasis in humans, mice and yeast. This is one <strong>of</strong> <strong>the</strong> projects in <strong>the</strong> field <strong>of</strong><br />
functional genomics (GEN-AU, GENome research in AUstria) funded by <strong>the</strong> Austrian<br />
Federal Ministry for Education, Science and Culture (bm:bwk).<br />
Activity-based enzyme stain<br />
Total protein stain<br />
The lipolytic proteome <strong>of</strong><br />
mouse adipose tissue<br />
Proteins <strong>of</strong> brown adipose tissue from fasted wild-type mice were labelled with a<br />
fluorescent lipase inhibitor followed by 2-D electrophoretic separation. The<br />
fluorescently tagged enzymes were detected with a laser scanner. A total protein stain<br />
(Sypro rubyTM) is shown as a control. Fluorescent spots were cut out and after tryptic<br />
28
in gel-digest, <strong>the</strong> isolated peptides were analyzed by nanoHPLC-MS/MS. So far, <strong>the</strong><br />
entire lipolytic proteome <strong>of</strong> mouse liver has also been determined. The function <strong>of</strong><br />
overexpressed enzyme candidates is currently being identified using defined lipid<br />
substrates<br />
2.2. Protein microarrays – Enzyme chips<br />
In <strong>the</strong> framework <strong>of</strong> <strong>the</strong> Kplus Research Centre<br />
Applied Biocatalysis (<strong>TU</strong> <strong>Graz</strong>), we develop<br />
"biochips" representing ordered microarrays <strong>of</strong><br />
biospecific ligands for <strong>the</strong> detection and<br />
quantification <strong>of</strong> lipolytic enzymes in biological<br />
samples. In addition, microarrays <strong>of</strong> enzymes are<br />
generated to determine protein affinity for substrate<br />
analogs. The development <strong>of</strong> <strong>the</strong>se tools basically<br />
requires three steps, namely specific loading <strong>of</strong><br />
activated supports with bioaffinity ligands,<br />
measurement <strong>of</strong> lipase binding (e.g. by<br />
fluorescence techniques), and application <strong>of</strong> <strong>the</strong><br />
Angewandte<br />
Biokatalyse<br />
Kompetenzzentrum GmbH<br />
bio chips to <strong>the</strong> investigation <strong>of</strong> real analytical problems. These systems will be used for <strong>the</strong><br />
screening <strong>of</strong> lipases with specific substrate acceptance.<br />
International Cooperations:<br />
T. Hianik, Department <strong>of</strong> Biophysics and Chemical Physics, Comenius University,<br />
Bratislava, Slovakia<br />
P. Deigner, SIRS Lab, Jena, Germany<br />
E. Lacôte, Chargé de Recherche CNRS, Université Pierre et Marie Curie, Paris, France<br />
C. Ferreri, I.S.O.F. - BioFreeRadicals , Consiglio Nazionale delle Ricerche, Bologna<br />
Italy<br />
C. Tringali, Dipartimento di Scienze Chimiche, Universita di Catania, Italy<br />
D. Russell, Department <strong>of</strong> Microbiology & Immunology, College <strong>of</strong> Veterinary<br />
Medicine<br />
Cornell University, Ithaca, USA<br />
Diploma <strong>the</strong>sis completed:<br />
Peter Krempl: Development <strong>of</strong> an electrophoresis method for differential analysis <strong>of</strong><br />
lipolytic enzymes<br />
In functional proteomics, "activity-based probes" (ABPs) are essential tools for labelling<br />
and detection <strong>of</strong> biocatalysts. Typical ABPs contain a specific binding group, a reactive<br />
functional moiety and a reporter group (e.g. a fluorescent dye) for protein detection.<br />
This study describes <strong>the</strong> chemical syn<strong>the</strong>sis and application <strong>of</strong> a set <strong>of</strong> matched ABPs<br />
for detection <strong>of</strong> lipases and esterases, that can be used for a precise comparative<br />
29
characterisation <strong>of</strong> several lipolytic enzymes in a complex proteome. For this purpose,<br />
proteome samples are exposed to <strong>the</strong> ABPs, followed by electrophoretic protein<br />
separation, acquisition and s<strong>of</strong>tware-assisted analysis <strong>of</strong> <strong>the</strong> fluorescence images <strong>of</strong> <strong>the</strong><br />
obtained protein patterns. The new method was validated using defined mixtures <strong>of</strong><br />
purified lipase and esterase preparations as well as natural tissue samples. The<br />
statistically valid thresholds for <strong>the</strong> detection <strong>of</strong> differences between <strong>the</strong> compared<br />
samples were determined.<br />
Research Projects:<br />
TIG Research center Applied Biocatalysis (coordinated by <strong>TU</strong> <strong>Graz</strong>): Novel lipases with<br />
specific substrate acceptance.<br />
bm:bwk (Genomforschung in Österreich, GEN-AU): Functional and differential<br />
analysis <strong>of</strong> lipolytic enzymes by affinity tagging (GOLD II project-Genomics <strong>of</strong><br />
Lipid-associated Disorders, coordinated by KFU <strong>Graz</strong>).<br />
FWF Doctoral Program Molecular Enzymology: Functional enzyme analysis<br />
(coordinated by KFU <strong>Graz</strong>).<br />
FWF Special Research Program (SFB) LIPOTOX (coordinated by KFU <strong>Graz</strong>): Toxicity<br />
<strong>of</strong> oxidized phospholipids in macrophages.<br />
Invited lectures:<br />
1) Effects <strong>of</strong> oxidized phospholipids on vascular smooth muscle cells. University <strong>of</strong><br />
Linz, Austria, March 6, 2007<br />
2) Novel fluorescent probes for lipids and lipases. 10 th International Conference on<br />
Methods and Applications <strong>of</strong> Fluorescence Spectroscopy, Imaging and Probes<br />
(MAF-10), Salzburg, Austria, 9 – 12 September 2007.<br />
3) Natürliche Antioxidantien in Lebensmitteln. Salzburger Gesundheitstage,<br />
Salzburg Austria, April 21, 2007.<br />
4) Apoptotic signaling <strong>of</strong> oxidized phospholipids in vascular smooth muscle cells<br />
and macrophages. Hermann Esterbauer Meeting, <strong>Graz</strong>, Austria, October 15, 2007<br />
5) Apoptotic effects <strong>of</strong> oxidized phospholipids on vascular smooth muscle cells.<br />
COST Meeting Bioradicals, Rome, Italy, September 14, 2007<br />
Publications:<br />
1) Moumtzi A, Trenker M, Flicker KH, Zenzmaier E, Saf R and Hermetter A<br />
Import and fate <strong>of</strong> fluorescent analogs <strong>of</strong> oxidized phospholipids in vascular<br />
smooth muscle cells<br />
J.Lipid Res. 48: 565 – 582, 2007<br />
2) Yates RM, Hermetter A, Taylor GA and Russell DG<br />
Macrophage activation down-regulates <strong>the</strong> degradative capacity <strong>of</strong> <strong>the</strong> phagosome<br />
Traffic 8: 241 – 250, 2007<br />
30
3) Wehinger A, Tancevski I, Schgoer W, Eller P, Hochegger K, Morak M, Hermetter<br />
A, Ritsch A, Patsch JR and Foeger B<br />
Phospholipid transfer protein augments apoptosis in THP-1-derived macrophages<br />
induced by lipolyzed hypertriglyceridemic plasma<br />
Arterioscler.Thromb.Vasc.biol. 27: 908 – 915, 2007<br />
4) Werner ER, Hermetter A, Prast H, Golderer G and Werner-Felmayer G<br />
Widespread occurrence <strong>of</strong> glyceryl e<strong>the</strong>r monooxygenase activity in rat tissues<br />
detected by a novel assay<br />
J.Lipid Res. 48: 1422 – 1427, 2007<br />
5) Tilz GP, Wiltgen M, Demel U, Faschinger C, Schmidinger H and Hermetter A<br />
Insights into molecular medicine: Development <strong>of</strong> new diagnostic and prognostic<br />
parameters<br />
Wien.Med.Wochenschr. 157: 122 – 129, 2007<br />
6) Fruhwirth GO, Loidl A and Hermetter A<br />
Oxidized phospholipids: From molecular properties to disease<br />
Biochim.Biophys.Acta 1772: 718 – 736, 2007<br />
7) Birner-Gruenberger R and Hermetter A<br />
Activity-based proteomics <strong>of</strong> lipolytic enzymes<br />
Curr.DrugDiscov.Technol. 4: 1 – 11, 2007<br />
8) Fruhwirth GO and Hermetter A<br />
Seeds and oil <strong>of</strong> <strong>the</strong> Styrian oil pumpkin: Components and biological activities<br />
Eur.J.LipidSci.Technol. 109: 1128 – 1140, 2007<br />
31
Lectures and Laboratory Courses<br />
LU aus Biochemie I<br />
5,33 LU <strong>Staff</strong><br />
Biochemie II 1,5 VO Macheroux P<br />
LU aus Biochemie II 4 LU <strong>Staff</strong><br />
Bachelorarbeit 1 SE Daum G<br />
Seminar zur Masterarbeit aus Biochemie und<br />
Molekularer Biomedizin<br />
2 SE Daum G, Hermetter<br />
A, Macheroux P<br />
Anleitung zu wissenschaftlichen Arbeiten aus<br />
Biochemie und Molekularer Biomedizin<br />
2 SE Daum G, Hermetter<br />
A, Macheroux P<br />
Molekulare Physiologie 2 VO Macheroux P<br />
Projektlabor Bioanalytik 6 PR Daum G, Hermetter<br />
A, Macheroux P<br />
Molekulare Enzymologie I 3 PV Macheroux P<br />
GL Chemie (BMT) 2 VO Hermetter A<br />
Membran Biophysik 3 PV Lohner K<br />
Membran-Mimetik 1 VO Lohner K<br />
Biophysikalische Chemie 3 PV Laggner P<br />
Biophysikalische Chemie der Lipide 3 PV Hermetter A<br />
Zellbiologie der Lipide 3 PV Daum G<br />
Zellbiologie 1 1 SE Daum G<br />
Physik.Meth.i.d.Biochemie 2 LU Laggner P<br />
Physikalische Methoden in der Biochemie 1 VO Laggner P<br />
Biochemie I<br />
3,75 VO Macheroux P<br />
LU aus Molekularbiologie 3 LU A<strong>the</strong>nstaedt K<br />
Seminar zu den LU aus Molekularbiologie 1 SE A<strong>the</strong>nstaedt K<br />
Immunologische Methoden 2 VO Daum G<br />
Immunologische Methoden 2 LU Daum G<br />
Bachelorarbeit 1 SE Daum G<br />
Seminar zur Masterarbeit aus Biochemie und<br />
Molekularer Biomedizin<br />
2 SE Daum G, Hermetter<br />
A, Macheroux P<br />
Anleitung zu wissenschaftlichen Arbeiten aus<br />
Biochemie und Molekularer Biomedizin<br />
2 SE Daum G, Hermetter<br />
A, Macheroux P<br />
Spezielle Kapitel der Biochemie 1 VO Daum G, Hermetter<br />
A, Macheroux P<br />
Fluoreszenztechnologie 2 VO Hermetter A<br />
Fluoreszenztechnologie 1,5 LU Hermetter A<br />
Molekulare Enzymologie 2 VO Gruber K, Macheroux<br />
P, Nidetzky B<br />
Projektlabor Bioanalytik 6 PR Daum G, Hermetter<br />
A, Macheroux P<br />
Molekulare Enzymologie II 3 PV Macheroux P<br />
GL Biochemie (BMT) 2 VO Macheroux P<br />
Membran Biophysik 3 PV Lohner K<br />
Biophysikalische Chemie 3 PV Laggner P<br />
Biophysikalische Chemie der Lipide 3 PV Hermetter A<br />
Zellbiologie der Lipide 3 PV Daum G<br />
Strukturanalyse in Biophysik u. Materialforschung 2 VO Laggner P<br />
Zellbiologie 2 1 SE Daum G<br />
Immunologische Methoden 1 VO Daum G<br />
32