Identification of /Fishing out binding partners from a protein complex containing OPA1 which is responsible for neuro-ophthamological disorder-OPTIC ATROPHY

Akepati V.R., Bulczak A., Alexander C.

Max-Delbrück-Center for Molecular Medicine, Neurodegeneration, Berlin, Germany

OPA1, a member of the dynamin protein family of large GTPases that are involved in

the basic events of membrane fusion and fission, is a mitochondrial protein. Apart from the

obvious common feature of these dynamin-related proteins, their GTPase domain, OPA1 has

two predicted coiled coil domains and mitochondrial leader sequence. OPA1 is localized to

mitochondrial intermembrane space, where it is tightly bound to outer leaflet of the inner

membrane. Mutations in the OPA1 gene cause autosomal dominant optic atrophy (ADOA), a

neuro-ophthalmological disorder leading to blindness in humans due to degeneration of the

optic nerve. Estimated disease prevalence is between 1:10,000 in Denmark and 1:50,000

worldwide.

The aim of our study is to biochemically characterize the OPA1 protein, emphasizing

majorly on revealing whether it exits as a component of a protein complex and which

proteins it interacts with. To achieve this goal, we are applying proteomics techniques. Blue

native gel electrophoresis is being used to identify and purify the OPA1 complex in its native

state and to determine its molecular weight. In order to identify potential interaction partners,

our first approach was to perform a high-throughput yeast two-hybrid screen for various

proteins expressed in the brain. In the second approach we are immunoprecipitating OPA1

from isolated mouse brain mitochondria. Subsequently, the identity of co-precipitated

proteins will be revealed by mass-spectrometry. Finally, based on the OPA1 interacting

proteins the functional relevance of this complex for the neuronal cell and, in particular,

retinal ganglion cells shall be investigated.

Open tubular capillaries for pre-concentration and Micro-extraction of

phosphopeptides

R. Bakry, G.K. Bonn

Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens

University, Innrain 52a, 6020-Innsbruck, Austria

The study of protein phosphorylation has grown exponentially in recent

years as researchers from various disciplines have come to realize that key

cellular functions are regulated by protein phosphorylation. The use of

immobilized metal affinity chromatography (IMAC) to separate phosphor-

peptides from peptide mixtures has been shown to be useful prior to both matrix

assisted laser desorption ionisation time of flight (Maldi-Tof) and electrospray

ionisation (ESI) mass spectrometry (MS) because suppression effects or low

average charge can be greatly reduced due to the presence of doubly charged

phosphate groups combined with the positively charged amino groups.

We introduce an open tubular capillary, where the inner wall of a fused

silica capillary is modified through covalent bonding with modified polystyrene

latex particles. The particles were derivatized with iminodiactic acid and

chelated with Fe+3 for the purification and enrichment of phosphopeptides. The

capillaries were successfully applied for selective pre-concentration of

phosphorylated peptides with recoveries ranging from 92-95 %, using a β-casein

tryptic digest and ovalbumin tryptic digest. The eluted components were applied

directly to a Maldi target for identification without any desalting step.

Studying γ-Protocadherins

Bonn S.

Max Planck Institute for medical research

Molecular Neurobiology

Jahnstrasse 29

69120 Heidelberg

γ-Protocadherins are type I membrane spanning glycoproteins. Arranged in tandem, the

protocadherin gamma locus contains 22 functional variable exons, each encoding extracellular

and transmembrane sequences, and three exons for an intracellular invariant domain.

γ-Protocadherins are subject to regulated intramembrane proteolysis (RIP), releasing the

intracellular domain (γICD) which subsequently translocates to the nucleus. How the

intracellular domain is translocated to the nucleus and the biological significance of this is to

date elusive.

My interest lies in elucidating interaction partners and post translational modifications of the

γICD, casting light on gamma Protocadherins mysterious biological function.

Optimization of in-gel digest conditions of ABC-transporter P-glycoprotein 1Brokemper O. 1Bücker A., 2Meyer J., 2Wiese M., 1Zabe-Kühn M. 1Functional Peptides, Research center caesar, Bonn; 2Pharmaceutical Institute, University of Bonn Overexpression of the ABC-transporter P-glycoprotein (P-gp) in tumor cells leads to a therapy resistance towards cytostatic drugs, a symptom called multidrug resistance. A so far unresolved phenomenon is the structural diversity of the molecules transported across the cell membrane by P-gp, a 190kDa membrane protein. With molecular modelling methods two binding sites of several ligands have been postulated. Identification of relevant substrate binding sites is a crucial step in understanding the broad substrate recognition by P-gp and possibly of similar transporters, which might lead to the development of new inhibitors of P-gp for chemotherapeutic approaches in the fight against tumors. In this project we aim at verifying the postulated binding sites by covalently binding photoactivable derivatives of known ligands to P-gp. We will then purify the labelled protein with SDS-PAGE, digest it with different proteases and search for peptides modified by the labelling process with mass spectrometric methods (LC-ESI-MS, LC-MALDI). The first steps are the optimization of the digest conditions for P-gp and a hight sequence coverage by use of different detergents and combined use of ESI- and MALDI-MS.

Quantifying Histone Posttranslational Modification in Schizosaccharomyces pombe using Mass Spectrometry. Buchanan LN, Roguev AB, Stewart AF. BIOTEC, Technische Universitaet Dresden, Tatzberg 47-51, 01307 Dresden, Germany Histone posttranslational modification (PTM) is a crucial factor in the control of higher order chromatin structure and transcriptional regulation. Bioinformatics reveals a multitude of genes in the Schizosaccharomyces pombe genome encoding specific protein domains implicated in histone PTM and other chromatin functions. Of particular interest are SET, CHROMO, SPRY and JmjC domains. All are present in chromatin-related proteins, although each performs a variety of functions. Which of these domains and which of the proteins that contain these domains are responsible for histone PTM? To investigate this question we are using mass spectrometry to detect and quantify global levels of histone PTM in S. pombe. Histones are obtained by acid extraction followed by HPLC purification. Both intact and digested histones are analysed by LC/MS/MS. By comparing quantities of histone PTMs between wild type yeast and strains harbouring mutations in genes of interest, it is possible to gain direct insight into whether these proteins are functioning in histone PTM. In order to make accurate comparisons between mutants and wild type yeast, precise quantification of modified histone variants is essential and may require isotopic labeling of purified histones. As an initial screen this MS analysis may identify proteins involved in histone PTM. It can also be used to quantitatively characterize proteins previously identified as histone modifiers. One limitation of this assay is that only global levels of histone PTM are measured. Region specific effects on histone PTM may be undetectable due to insignificant change in global levels.

A Systems Biology approach for the characterization of metabolic bottlenecks in recombinant protein production processes

Carneiro, S., Rocha, I. and Ferreira, E. C. Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057

Braga, Portugal The purpose of this project is to derive strategies for increasing the productivity of recombinant protein production processes by applying a systems biology perspective to the phenomena occurring in the recombinant cell. This will specifically involve the use of genome-scale analysis of the transcriptome, proteome and fluxome. Escherichia coli have been the organism of choice for the production of many recombinant proteins with high therapeutic value. However, there are still some associated phenomena that decrease the process performance, like the stringent response that usually occurs when very high levels of heterologous protein production. In this work, the high-cell density fed-batch recombinant protein production process in E. coli will be studied, giving particular relevance to stringent response. The approach is intended to be systematic, by first compiling the existing knowledge about this phenomenon, extending existing genome-scale models to accommodate that knowledge, and derive hypothesis in silico that will then be tested by using genome-scale analysis of the transcriptome.

Analysis of differently expressed proteins in synaptosomes isolated from PrPC knockout mouse brains

1Crecelius, A.; 1Helmstetter, D.; 1Wünsch, G.; 2Fröhlich, T.; 2Arnold, G.; and

1Kretzschmar, H.

1Centre for Neuropathology and Prion Research, Ludwig-Maximilians-University Munich, Feodor-

Lynen-Str. 23, 81377 Munich. 2Gene Center, LAFUGA, Ludwig-Maximilians-University Munich, Feodor-Lynen-Str. 25, 81377

Munich.

Prion diseases are a class of neurodegenerative disorders affecting both humans

and animals. Their pathogenesis is associated with the conversion of the prion

protein (PrPC) to the infectious isoform (PrPSc). The aim of this study is to identify

differently expressed synaptosomal proteins correlated to the loss of PrPC for a

better understanding of its function. Synaptosomes are artificial structures of

pinched-off nerve endings with vesicles, cytoplasm, and the attached

subsynaptic areas of the postsynaptic cell membranes. The proteome analysis

of synaptosomes compared to whole brain homogenates has the advantage of

reducing the sample complexity and enriching proteins associated with synaptic

function. Two wild-type and knockout mouse brains (male, eight weeks old)

respectively will be pooled to isolate the synaptosomes by several centrifugation

steps of the nervous tissue homogenate. Proteomic analysis will be performed

on five independent synaptosomal preparations of each mouse type to gain

statistical confidence of protein expression differences. Both quantitative

proteomic techniques, 2-D DIGE combined with MALDI-MS/MS identification of

the digested 2-D spots and LC-MS based ICAT analysis will be employed for

pair-wise comparison of the synaptosomal preparations.

Biogenesis and function of the thylakoid membrane system in higher plants Dal Bosco C., Herrmann R.G. and Meurer J. Ludwig-Maximilians-Universität, Department Biologie I, Botanik, Menzingerstr. 67, 80638 München, Germany Thylakoid membranes are specialised biomembranes capable of solar energy transformation. Our aim is to investigate the biogenesis as well as the structure/function relationship of the thylakoid membrane system consisting of four major multi subunit membrane complexes, which are of dual genetic origin. Therefore, a collection of knock-out plastome mutants in tobacco and nuclear mutants in Arabidopsis for mostly low molecular weight subunits of the photosystems II and I, the cytochrome bf complex as well as the ATP synthase is under study with regard to the kinetics of assembly processes and to functional aspects since their function is presently almost unknown. Interestingly, many of these mutants are still viable but distinct steps during assembly and specific functions of the complexes are affected in the individual lines. Future attempts mainly focus on proteomic approaches in combination with in vivo labeling of thylakoid membrane proteins to probe into the stepwise assembly of the complexes and the functional role of individual subunits. Recent work has uncovered that generation, maintenance and repair of the thylakoid system are highly sophisticated processes. Therefore, membrane dynamics like movement of proteins within the thylakoid membrane (state transition), the reversible dissociation of chlorophyll antenna from core complexes and the turn over of individual subunits will be investigated. For instance, the nuclear atpC1 gene encoding the gamma subunit of the plastid ATP synthase has been inactivated by T-DNA insertion mutagenesis in Arabidopsis thaliana. In the seedling-lethal mutant, the absence of detectable amounts of the gamma subunit destabilizes the entire ATP synthase complex. To investigate the above-mentioned assembly processes and the function of the rotating gamma subunit recombinant forms of the atpC gene were introduced into the mutant background to partially complement the phenotype.

PROTEOMIC APPROACH TO IDENTIFY THE SECRETOME OF A HUMAN COLON CARCINOMA CELL LINE VIA 2D-PAGE AND MALDI-TOF/TOF-MS Diehl H.1,2, Stühler K.1, Volmer M.W.2, Klein-Scory S.2, Schöneck A.2, Schmiegel W.2, Meyer H. E.1, Schwarte-Waldhoff I.2 1Medical Proteom-Center, 2Department of Internal Medicine (IMBL), Knappschafts-krankenhaus, Ruhr – University Bochum, Bochum, Germany The identification of novel protein biomarkers to improve cancer diagnosis is a major goal in cancer research. The direct detection of biomarkers in human serum is difficult due to the fact, that such proteins released by tumours in vivo occur at very low abundance. Arguing, that the proteins released by tumour cells in vitro, a subproteome tentatively termed the “secretome”, to a certain extent may reflect proteins released in vivo we started to assess tumour cell secretomes as an alternative source for possible cancer markers. As knowledge concerning the composition of such “secretomes” is limited, the aim of this study was to establish a catalogue of secretome proteins derived from SW620 human colon cancer cells using modern proteomic techniques. Proteins prepared from conditioned media under serum-free conditions, a prerequisite for the analysis, were concentrated and resolved by 2D-PAGE prior to visualizing the resulting spot pattern by silver-staining. A total of 355 protein spots were excised and in-gel digested with trypsin. The extracted peptides were analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Approximately two thirds were identified through database searches applying Profound and Sequest search algorithms. The putative origin of the identified secretome proteins was addressed by comparison with protein databases, published literature and through the analysis of leader sequences using the SigCleave program. The SW620 “secretome” is composed of proteins released through classical and non-classical secretion pathways as well as through the release of exosomes. The SW620 secretome catalogue combined with 2D gel analysis reveals new insights into the composition and amount of proteins released in vitro and can thus be used as a basis to decipher putative novel serum biomarkers. It also serves for comparison with secretomes derived from other (tumour) cells.

In vivo horizontal gene transfer from transplanted HUVEC into rat cardiomyocytes Goettsch S1, Ding Z1, Gödecke S1, Assmann A1, Bloch W2, Wirrwar A3, Buchholz D3, Antke C3, Leurs C4, Hanenberg H5, Müller H-W3, Schrader J1 1 Department of Physiology, Heinrich-Heine-University, Duesseldorf, Germany; 2 Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany; 3 Institute for Nuclear Medicine, Heinrich-Heine-University, Duesseldorf, Germany; 4 Qiagen GmbH, Hilden, Germany, 5 Clinic for Paediatric Oncology, Haematology and Immunology, Heinrich-Heine-University, Duesseldorf, Germany Human umbilical vein endothelial cells (HUVECs) are considered to be mature cells, but

were reported to transdifferentiate in vitro into smooth muscle cells and cardiomyocytes. To

test this concept in vivo we transplanted HUVECs, labelled with 111-Indium or EGFP, via the

coronary artery system into the rat heart using a double lumen balloon catheter. As

determined by SPECT intracoronary delivery of 111-Indium labelled HUVECs resulted in the

stable attachment of 18% of infused cells without alterations of cardiac function within 3

weeks. To track the long-term cell fate HUVECs stably expressing EGFP after lentiviral

transduction were used. One day after transplantation HUVECs stained positive for PECAM-

1 and human mitochondrial proteins, but were negative for rat MCT-1. After 3 and 7 days

EGFP expressing cells showed typical cardiomyocytic morphology and were positive for a-

actinin and rat MCT-1; however, they were negative for PECAM-1 and human mitochondrial

proteins. After 3 weeks no EGFP expressing cells were detected. Immunohistochemistry

revealed a transient activation of caspase-3 and a deactivation of Akt/PKB, suggesting

apoptosis of HUVECs in vivo. To explore whether the labelling of cardiomyocytes in vivo is

due to horizontal gene transfer initiated by apoptosis we isolated apoptotic bodies from the

supernatant of cultured EGFP expressing HUVECs followed by coculture with

cardiomyocytes. HUVECs-derived apoptotic bodies carried DNA-fragments coding for EGFP

as could be determined by PCR, but no EGFP protein. Coculture of neonatal rat

cardiomyocytes with apoptotic bodies from HUVECs for 5 days resulted in EGFP labelled

cardiomyocytes that were positive for myosin heavy chain.

This study reports the effective transplantation of HUVECs into the rat heart by intracoronary

cell delivery. While we did not find evidence for transdifferentiation of HUVECs in vivo, we

describe a substantial and selective uptake of HUVECs-derived DNA by cardiomyocytes

initiated by apoptosis.

Identification of virus receptors using a proteomics-based approach A. S. Hahn, B. Fleckenstein, F. Neipel Institut für klinische und molekulare Virologie, Universität Erlangen Specific interactions of viral envelope glycoproteins with cellular ligands are a first and crucial step in the viral life cycle. In the case of human herpesvirus-8 (HHV-8), three glycoproteins (K8.1, gH, gB) have been shown by us and other groups to bind to cell surface heparan sulfate. In addition, gB of HHV-8 has recently been shown to promote cell entry by interaction with certain integrins. Nevertheless, the role of HHV-8 surface proteins in virus attachment and entry is still poorly understood. A proteomics-based approach will be used to identify additional cellular receptors of HHV-8 and investigate interactions of HHV-8 surface glycoproteins with cellular ligands. For this purpose, we express viral envelope glycoproteins as so-called soluble “immunoadhesins”, chimeric proteins comprising the extracellular part of the viral surface proteins fused to the Fc-fragment of human IgG1. Various techniques including immunoprecipitation, far western blotting, in vivo crosslinking, affinity chromatography and 2-D electrophoresis are currently used to isolate cellular interaction partners, which will then be identified by peptide mass fingerprinting. Applying these techniques with HHV-8 gH-Fc as a probe we could already show that it interacts with at least two single-chain proteins of approximately 35 kDa and 70-90 kDa. Although initially obtained by Far Western blotting, we could already confirm the interactions by crosslinking studies and immunoprecipitation. Experiments to identify these two putative novel gH-ligands by mass spectrometry are currently ongoing. Identification of these cellular proteins will by followed by studies assessing their functional role in the HHV-8 life cycle. Once fully established in the laboratory, these techniques shall be adopted to a wider range of pathogens.

Surface Enhanced and Automated Protein Microarrays Hartmann M.1, Pabst S.1,Templin M. 1, Joos T. 1 and Roeraade J.2 1NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen, D-72770 Reutlingen, Germany and 2KTH Royal Institute of Technology, SE-100 44 Stockholm, Sweden Protein Microarrays allow the simultaneous analysis of thousands of molecules

extracted from minute samples of tissues or body fluids. This is done by applying

specific capture molecules as arrays of minispots on specially coated surfaces.

Analytes in the sample bind to their respective capture molecules and quantitative

analysis of binding parameters is performed by optical procedures.

The focus of the work presented here is to improve the performance of protein

microarrays using surface enhancements, implementation of surface acoustic wave

SAW mixing technology and automation of the assays.

Chemically and structurally-modified surfaces offer better possibilities for the

functional immobilization of biomolecules with the goal of increasing signal density

and to reduce unspecific binding.

SAW increase reproducibility, circumvents the limitations of diffusion,

Automation of the assays will increase the robustness and reliability of the assays.

Enhancing spotting techniques and read out systems will be integrated. Focus on

autoimmune

Using this appraoches, => resulting in an increase of signal/noise ratio

to achieve a better discrimination of low positive and negative samples.

In addition this set up can be applied to any other type of protein microarray

approach like like sandwich immunoassays or antigen – antibody interacitons.

The work is an integral part of the European Union project entitled

“Nanobiotechnology with Self-Organising Structures” (NABIS). The goal of the project

is to study and develop innovative technologies for the next generation of high

performance biochips, for future applications e.g. in accelerated drug discovery,

diagnostics and personalized medicine. Please visit www.nabis.kth.se for a more

detailed description.

Transcriptional regulation of tricarboxylic acid cycle genes from

Corynebacterium glutamicum Hasenbein, S., Bott, M.

Biochemistry Group, Institute of Biotechnology 1, Research Center Juelich, D-52425 Juelich,

Germany

Corynebacterium glutamicum, a non-pathogenic Gram-positive soil bacterium, is widely used

in the biotechnological production of various amino acids, such as L-glutamate and L-lysine.

The tricarboxylic acid cycle (TCA cycle) and its bypasses play important roles in the central

metabolism of C. glutamicum as they serve for energy supply as well as for provision of

biosynthetic precursors. Understanding the reactions and regulatory mechanisms of central

metabolism is therefore key to improve producer strains. Besides its use in industry, C.

glutamicum also represents a model organism of the Corynebacterineae, which includes the

pathogenic C. diphtheriae and Mycobacterium tuberculosis. Knowledge about the central

metabolism of C. glutamicum thus could also lead to new targets for antiinfectives.

The biochemical and genetic characteristics of several TCA cycle enzymes have been

studied in detail. However, until now only little information is available concerning the

genetic regulation of the corresponding genes and global regulatory mechanisms of central

metabolism. As demonstrated for many bacteria, expression of genes is often controlled at the

transcriptional level by DNA binding proteins. This mechanism of regulation has recently

been shown for the genes of the glyoxylate bypass and the aconitase gene in C. glutamicum.

In the future, we plan to search for additional transcriptional regulators of genes involved in

the central metabolism of C. glutamicum. By use of DNA affinity chromatography we want to

enrich transcriptional regulators binding to selected promoters. Identification of these proteins

will then be performed by peptide mass fingerprinting using MALDI-TOF based on the

complete C. glutamicum genome sequence. Subsequently, mutants lacking the genes encoding

the identified regulators will be constructed and compared to the wild type by transcriptome

and proteome analysis. In preliminary experiments, we could verify the binding of the TetR-

type repressor AcnR (Krug, Wendisch and Bott (2005) J. Biol. Chem. 280: 585-595) to the

promoter region of the aconitase gene acn, confirming the functionality of our approach.

Biomarker discovery in breast cancer serum using 2D-DIGE and mass spectrometry Hong-Lei Huang1, Taras Stasyk1, Sandra Morandell1, Isabel Feuerstein2, Christian W. Huck2, Günther Stecher2, Maurice Mogg3, Martin Schreiber3, Guenther K. Bonn2, Lukas A. Huber1 1 Biocenter, Division of Cell Biology, Innsbruck Medical University, Austria. 2 Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck. 3 Division of Special Gynecology, Medical University of Vienna Worldwide, breast cancer is the most frequent cancer in women. The discovery of biomarkers will facilitate the prevention, diagnosis and therapy of breast cancer. Since blood constantly perfuse cancer tissues, it might be expected that the onset or presence of disease can be determined by measuring the altered presence or abundance of the biomarkers in serum. In the present study, we used 2-D DIGE and Mass spectrometry strategy to screen biomarker candidates in serum samples. Firstly, we pooled the serum samples taken from patients and benign match with age and menopause. Then, we deplete the most high abundant proteins Albumin/IgG under partly denature conditions in order to enrich low abundant proteins and proteins with low molecular weight, 5% ACN was added for the purpose to release the low molecular weight proteins from the carriers protein albumin. After sample concentration and desalting, samples were labeled with three different dyes, and separated with 2D-DIGE. Nine high expressed proteins and eight low expressed proteins were detected in patient groups using biological variance analysis software package. Finally, all these candidates were picked and in gel digested with trypsin, MALDI-TOF/TOF was employed to identify these candidates. The function evaluation of these candidates were discussed at the end.

A Novel Strategy for Detection of Low Stoichiometry Protein Phosphorylation Sites by TiO2 Enrichment and nanoElectrospray MS/MS

- Application to Human Galectin Hung C.-W. 1, Kübler D. 2, Gabius H.-J. 3, Krüger R. 1, Schlosser A. 4, Lehmann W. D.1 1Central Spectroscopy and 2Department of Pathochemistry German Cancer Research Center (DKFZ), Heidelberg, Germany 3 Institute of Physiological Chemistry, LMU University Munich, Germany 4 Institute of Medical Immunology, Charité, Berlin

Galectins represent a family of widely distributed multifunctional carbohydrate-

binding proteins, for which various qualitative and quantitative changes have been

demonstrated to occur in the development and outcome of cancer [1]. Reversible protein

phosphorylation is one of the most important post-translational modifications. It is assumed

that phosphorylation also plays a key role in the functional regulation of galectins but so far

only few sites could be identified.

We have set up a novel analytical strategy for analysis of protein phosphorylation sites

with low phosphorylation stoichiometry consisting of the combination of in-gel protein

digestion with a combination of trypsin and AspN, phosphopeptide enrichment on a

microcolumn filled with titanium dioxide [2,3] and manual or automated nanoelectrospray

tandem mass spectrometry. Using ovalbumin and β-casein as model phosphoproteins, the

TiO2 extraction efficiency for phosphopeptides of 1-2.5 kDa size was measured to be about 80

% using element mass spectrometry. Peptides containing His, Trp, or multiple residues of Asp

and Glu were observed to be coextrated with a certain efficiency, but phosphopeptides are

safely recognized on the basis of their unique MS/MS spectra. The prolonged data acquisition

times that can be realized using nanoESI MS/MS provide the possibility to acquire tandem

MS spectra of outstanding quality. Finally we have applied this novel methodology to

recombinant human galectin after its in vitro phosphorylation and have successfully identified

two previously unknown phosphorylation sites.

References: [1] Liu FT, Rabinovich GA. Nature Reviews Cancer 2005, 5, 29-41. [2] Sano A, et al. Anal. Sci. 2004, 20, 565-566. [3] Pinkse MWH, et al. Anal. Chem. 2004, 76, 3935-3943.

Inhibition of ribonucleotide reduction and cell cycle arrest induced by manganese depletion in the gram-positive bacterium Bacillus subtilis Nguyen, Huyen, and Auling, Georg Institute of Microbiology, University, Schneiderberg 50, D-30167 Hannover, Germany Ribonucleotide reduction, a unique step in the route to DNA, is a manganese-dependent reaction in the gram-positive prokaryote Bacillus subtilis (Mohamed et al., BioFactors 7:337-344, 1998). A proteomic study using the DIGE-technique revealed more than hundred of two thousend proteins which appear to be regulated by manganese depletion during growth in pyruvate-mineral medium. Cells of B. subtilis responded to manganese depletion by phenomena of unbalanced growth death like inability to synthesize DNA, cell cycle arrest and filamentation while the majority of proteins was up-regulated. Future studies are necessary to compare this gene regulation with known phenomena of DNA-damage (SOS-induction)

Proteomics in Schizosaccharomyces pombe;

Analysis of 11-deoxycorticosterone (DOC) and aldosterone

dependent differential protein patterns Kyung Hoon Hwang1, Susanne Böhmer1, Christine Carapito2, Emmanuelle Leize2,

Alain Van Dorsselaer2, Rita Bernhardt1

1 Universität des Saarlandes, FR 8.3 Biochemie, P.O. Box 151150, D-66041 Saarbrücken, Germany 2 Laboratoire de Spectrométrie de Masse Bio-Organique, ECPM, 25, rue Becquerel, UMR 7509-

CNRS/Université Louis Pasteur, Strasbourg, France

11-deoxycorticosterone (DOC) is an aldosterone precursor that has anticonvulsant

properties in animals and possibly also in humans. Aldosterone is the most important

mineralocorticoid in the human body. Mineralocorticoids play a major role in regulating

sodium (Na+) and potassium (K+) homeostasis and also contribute to the control of blood

pressure and to some physiological disorders. Very recently, it has been found that high

aldosterone levels are not only linked to hypertension, but also play a role in the development

of congestive heart failure. The mechanisms of these changes are poorly understood yet. The

fisson yeast Schizosaccharomyces pombe is an unicellular eucaryote and contains many genes

and regulatory mechanisms, which are close to those of mammals. It thus represents a simple

model for investigating complex mammalian physiological and pathological interconnections.

The sequencing of the fission yeast genome was completed in 2002. In this study, we succeed

in 2D gels with a high resolution and high reproducibility. We have visualized more than

1500 protein spots on silver stained gel and found differentially expressed proteines by using

PDQuest software.. Proteins were identified by MALDI-TOF MS or a capillary LC Q-TOF II

in combination with Mascot search program. In total, so far 261 proteins were identified and

functionally classified on the bases of annotations from Swiss-Prot and TrEMBL, and S.

pombe database at Sanger Institute. The identified proteins represented 123 genes. Among the

261 proteins identified, 16 proteins (8 of which were down regulated, 8 of which were up

regulated.) were differentially regulated by DOC and were specifically associated with amino

acid biosynthesis, carbohydrate metabolism, glycolysis and gluconeogenesis, heat shock

proteins, ion transport, oxygen and radical detoxification, translation, or actin cytoskeleton

organization and biogenesis. We also found that several proteins were differentially regulated

by aldosterone. These proteins might be new targets for the development of drugs against

aldosterone-caused heart disease.