Hiltunen JK, Wenzel B, Beyer A, Erdmann R, Fosså A, Kunau WH.
Abteilung für Zellbiochemie, Medizinische Fakultät, Ruhr-Universität Bochum, Federal Republic of Germany.
J Biol Chem. 1992 Apr 5;267(10):6646-53.
The gene encoding the multifunctional protein (MFP) of peroxisomal beta-oxidation in Saccharomyces cerevisiae was isolated from a genomic library via functional complementation of a fox2 mutant strain. The open reading frame consists of 2700 base pairs encoding a protein of 900 amino acids. The predicted molecular weight (98,759) is in close agreement with that of the isolated polypeptide (96,000). Analysis of the deduced amino acid sequence revealed similarity to the MFPs of two other fungi but not to that of rat peroxisomes or the multifunctional subunit of the Escherichia coli beta-oxidation complex. The FOX2 gene was overexpressed from a multicopy vector (YEp352) in S. cerevisiae and the gene product purified to apparent homogeneity. A truncated version of MFP lacking 271 carboxyl-terminal amino acids was also overexpressed and purified. Experiments to study the enzymatic properties of the wild-type MFP demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA). The truncated form contained only the D-3-hydroxyacyl-CoA dehydrogenase activity. These results clearly demonstrate that the beta-oxidation of fatty acids in S. cerevisiae follows a previously unknown stereochemical course, namely it occurs via a D-3-hydroxyacyl-CoA intermediate.
Fosså A, Beyer A, Pfitzner E, Wenzel B, Kunau WH.
Institut fur Physiologische Chemie, Abteilung fur Zellbiochemie, Ruhr-Universität Bochum, Germany.
Mol Gen Genet. 1995 Apr 10;247(1):95-104.
We present the molecular cloning and sequencing of genomic and cDNA clones of the fox-2 gene of Neurospora crassa, encoding the multifunctional beta-oxidation protein (MFP). The coding region of the fox-2 gene is interrupted by three introns, one of which appears to be inefficiently spliced out. The encoded protein comprises 894 amino acid residues and exhibits 45% and 47% sequence identity with the MFPs of Candida tropicalis and Saccharomyces cerevisiae, respectively. Sequence analysis identifies three regions of the fungal MFPs that are highly conserved. These regions are separated by two segments that resemble linkers between domains of other MFPs, suggesting a three-domain structure. The first and second conserved regions of each MFP are homologous to each other and to members of the short-chain alcohol dehydrogenase family. We discuss these homologies in view of recent findings that fungal MFPs contain enoyl-CoA hydratase 2 and D-3-hydroxyacyl-CoA dehydrogenase activities, converting trans-2-enoyl-CoA via D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA. In contrast to its counterparts in yeasts, the Neurospora MFP does not have a C-terminal sequence resembling the SKL motif involved in protein targeting to microbodies.
Erdmann R, Wiebel FF, Flessau A, Rytka J, Beyer A,
Fröhlich KU, Kunau WH.
Abteilung fur Zellbiochemie, Ruhr-Universität Bochum, Federal Republic of Germany.
Cell. 1991 Feb 8;64(3):499-510.
PAS genes are required for peroxisome biogenesis in the yeast S. cerevisiae. Here we describe the cloning, sequencing, and characterization of the PAS1 gene. Its gene product, Pas1p, has been identified as a rather hydrophilic 117 kd polypeptide. The predicted Pas1p sequence contains two putative ATP-binding sites and reveals a structural relationship to three other groups of proteins associated with different biological processes such as vesicle-mediated protein transport (NSF and Sec18p), control of cell cycle (Cdc48p, VCP, and p97-ATPase), and modulation of gene expression of the human immunodeficiency virus (TBP-1). The proteins share a highly conserved domain of about 185 amino acids including a consensus sequence for ATP binding. We suggest that these proteins are members of a novel family of putative ATPases and may be descendants of one common ancestor.
Kunau WH, Beyer A, Franken T, Götte K, Marzioch M,
Saidowsky J, Skaletz-Rorowski A, Wiebel FF.
Institut fur Physiologische Chemie, Medizinische Fakultät, Ruhr-Universität Bochum, Germany.
In order to investigate the mechanisms of peroxisome biogenesis and to identify components of the peroxisomal import machinery we studied these processes in the yeast Saccharomyces cerevisiae. The forward genetic approach has led to pas-mutants (peroxisomal assembly) which fall into 12 complementation groups and allowed to identify 10 of the corresponding wild-type PAS genes (PAS 1-7, 9, 11 and 12). Recent sequence analysis data of some of these genes are beginning to provide first hints as to the possible function of their gene products. The PAS genes and their corresponding mutants are presently used to address some important questions of peroxisomal biogenesis. Reversed genetics has been started as a complementary approach to characterize especially the function of peroxisomal membrane proteins. For this purpose we describe a technique to isolate highly purified peroxisomes. This led to the identification of 21 polypeptides as constituents of this organelle. Some of them are presently sequenced.
Portsteffen H, Beyer A, Becker E, Epplen C, Pawlak A, Kunau WH, Dodt G.
Abteilung fur Zellbiochemie, Ruhr-Universität Bochum, Germany.
Nat Genet. 1997 Dec;17(4):449-52.
Human peroxisome biogenesis disorders (PBDs) are a group of genetically heterogeneous autosomal-recessive disease caused by mutations in PEX genes that encode peroxins, proteins required for peroxisome biogenesis. These lethal diseases include Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum's disease (IRD), three phenotypes now thought to represent a continuum of clinical features that are most severe in ZS, milder in NALD and least severe in IRD2. At least eleven PBD complementation groups have been identified by somatic-cell hybridization analysis compared to the eighteen PEX complementation groups that have been found in yeast. We have cloned the human PEX1 gene encoding a 147-kD member of the AAA protein family (ATPases associated with diverse cellular activities), which is the putative orthologue of Saccharomyces cerevisiae Pex1p (ScPex1p). Human PEX1 has been identified by computer-based 'homology probing' using the ScPex1p sequence to screen databases of expressed sequence tags (dbEST) for human cDNA clones. Expression of PEX1 rescued the cells from the biogenesis defect in human fibroblasts of complementation group 1 (CG1), the largest PBD complementation group. We show that PEX1 is mutated in CG1 patients.
Institut fur Physiologische Chemie, Medizinische Fakultät, Ruhr-Universität, Bochum, Germany. email@example.com
Protein Sci. 1997 Oct;6(10):2043-58.
The AAA protein family, a recently recognized group of Walker-type ATPases, has been subjected to an extensive sequence analysis. Multiple sequence alignments revealed the existence of a region of sequence similarity, the so-called AAA cassette. The borders of this cassette were localized and within it, three boxes of a high degree of conservation were identified. Two of these boxes could be assigned to substantial parts of the ATP binding site (namely, to Walker motifs A and B); the third may be a portion of the catalytic center. Phylogenetic trees were calculated to obtain insights into the evolutionary history of the family. Subfamilies with varying degrees of intra-relatedness could be discriminated; these relationships are also supported by analysis of sequences outside the canonical AAA boxes: within the cassette are regions that are strongly conserved within each subfamily, whereas little or even no similarity between different subfamilies can be observed. These regions are well suited to define fingerprints for subfamilies. A secondary structure prediction utilizing all available sequence information was performed and the result was fitted to the general 3D structure of a Walker A/GTPase. The agreement was unexpectedly high and strongly supports the conclusion that the AAA family belongs to the Walker superfamily of A/GTPases.
Beyer A, Kitzerow A, Crute B, Kemp BE, Witters LA,
Heilmeyer LM Jr.
Institut fur Physiologische Chemie, Ruhr-Universität Bochum, Germany.
Biol Chem. 2000 May-Jun;381(5-6):457-61.
AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (cAMPK) have been reported to phosphorylate sites on phosphorylase kinase (PhK). Their target residues Ser 1018 and Ser 1020, respectively, are located in the so-called multi- phosphorylation domain in the PhK alpha subunit. In PhK preparations, only one of these serines is phosphorylated, but never both of them. The aim of this study was to determine whether phosphorylation by cAMPK or AMPK would influence subsequent phosphorylation by the other kinase. Surprisingly, employing four different PhK substrates, it could be demonstrated that, in contradiction to previous reports, PhK is not phosphorylated by AMPK.
Schmitz FJ, Beyer A, Charpentier E, Normark BH, Schade M,
Fluit AC, Hafner D, Novak R.
Eijkman Winkler Institute for Medical Microbiology, Infectious Diseases and Inflammation, University Hospital Utrecht, Utrecht, The Netherlands.
J Infect Dis. 2003 Nov 15;188(10):1578-86. Epub 2003 Nov 05.
We determined the toxin-gene profiles of 239 endemic, invasive group A streptococcal (GAS) isolates that circulated, within a 5-year period, in European university hospitals. Profiling was performed by use of multiplex polymerase chain reaction that screened for 9 streptococcal pyrogenic exotoxins (speA, speB, speC, speF, speG, speH, speJ, ssa, and smeZ). Analysis revealed that invasive GAS isolates do not share a common toxin-gene profile. Although all emm types were characterized by several different toxin-gene profiles, a predominance of 1 or 2 toxin-gene profiles could be observed, reflecting that a few invasive clones have spread successfully throughout the world. Remarkably, statistical pair-wise analysis of individual toxin genes revealed that strains that did not share the predominant profile still showed a nonrandom distribution of key toxin genes characteristic of the specific emm type. This could indicate that M proteins function, directly or indirectly, as barriers for horizontal gene exchange.
Scheuring S, Bodor O, Röhricht RA, Müller S,
Beyer A, Köhrer K.
Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität Düsseldorf, Moorenstrasse 5, 40225, Düsseldorf, Germany.
Gene. 1999 Jun 24;234(1):149-59.
The mouse SKD1 protein displays a high degree of sequence identity (62%) to the yeast Vps4 protein, which is involved in the transport of proteins out of a prevacuolar/endosomal compartment. We isolated the mouse SKD1 locus and found that the SKD1 gene is split into 11 exons covering a region of 29kb of the genome. Interestingly, the exon/intron structure reflects to a certain degree the proposed domain structure of the protein, since the 5' located coiled-coil region and the AAA domain are flanked by introns. Analysis of the promoter region, which revealed features common for 'housekeeping genes', is consistent with previous results of a mouse multi-tissue Northern blot, confirming that SKD1 is a ubiquitously expressed gene. Expression of the full-length SKD1 cDNA in a vps4 disrupted yeast strain suppressed the temperature-sensitive growth defect of the vps4 mutant strain. Overexpression of wild type and expression of mutant Vps4 and SKD1 proteins, harbouring single amino acid exchanges in their AAA domains, induced a dominant-negative vacuolar protein sorting defect in wild type yeast cells, indicating that mouse SKD1 protein and yeast Vps4p fulfil similar functions.
Scheuring S, Röhricht RA, Schöning-Burkhardt B,
Beyer A, Müller S, Abts HF, Köhrer K.
Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität Düsseldorf, Moorenstrasse 5, Düsseldorf, D-40225, Germany.
J Mol Biol. 2001 Sep 21;312(3):469-80.
The yeast Vps4 protein (Vps4p) is a member of the AAA protein family (ATPases associated with diverse cellular activities) and a key player in the transport of proteins out of a prevacuolar endosomal compartment. In human cells, we identified two non-allelic orthologous proteins (VPS4-A and VPS4-B) of yeast Vps4p. The human VPS4-A and VPS4-B proteins display a high degree of sequence identity to each other (80 %) and to the yeast Vps4 protein (59 and 60 %, respectively). Yeast cells lacking a functional VPS4 gene exhibit a temperature-sensitive growth defect and mislocalise a carboxypeptidase Y-invertase fusion protein to the cell surface. Heterologous expression of human VPS4 genes in vps4 mutant yeast strains led, in the case of human VPS4-A, to a partial and, in the case of human VPS4-B, to a complete suppression of the temperature-sensitive growth defect. The vacuolar protein sorting defect of vps4 mutant yeast cells was complemented completely by heterologous expressed human VPS4-B protein, and partially by the human VPS4-A protein. Expression of mutant human VPS4-A (E228Q) and VPS4-B (E235Q) proteins, harbouring single amino acid exchanges in their AAA domains, induced dominant-negative vacuolar protein sorting defects in wild-type yeast cells in both cases. Two-hybrid experiments suggest that the human VPS4-A and VPS4-B proteins can form heteromeric complexes, and subcellular localisation experiments indicate that both human VPS4 proteins associate with endosomal compartments in yeast. Based on these results, we conclude that both human VPS4 proteins are involved in intracellular protein trafficking, presumably at a late endosomal protein transport step, similar to the Vps4p in yeast.
Beyer A, Scheuring S, Müller S, Mincheva A, Lichter P,
Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität Düsseldorf, Moorenstrasse 5, D-40225 Duesseldorf, Germany.
Gene. 2003 Feb 13;305(1):47-59.
The VPS4 gene is a member of the AAA-family; it codes for an ATPase which is involved in lysosomal/endosomal membrane trafficking. VPS4 genes are present in virtually all eukaryotes. Exhaustive data mining of all available genomic databases from completely or partially sequenced organisms revealed the existence of up to three paralogues, VPS4a, -b, and -c. Whereas in the genome of lower eukaryotes like yeast only one VPS4 representative is present, we found that mammals harbour two paralogues, VPS4a and VPS4b. Most interestingly, the Fugu fish contains a third VPS4 paralogue (VPS4c). Sequence comparison of the three VPS4 paralogues indicates that the Fugu VPS4c displays sequence features intermediate between VPS4a and VPS4b. Using complete mammalian VPS4a and VPS4b cDNA clones as probes, genomic clones of both VPS4 paralogues in human and mouse were identified and sequenced. The chromosomal loci of all four VPS4 genes were determined by independent methods. A BLAST search of the human genome database with the human VPS4A sequence yielded a double match, most likely due to a faulty assembly of sequence contigs in the human draft sequence. Fluorescent in situ hybridization and radiation hybrid analyses demonstrated that human and mouse VPS4A/a and VPS4B/b are located on syntenic chromosomal regions. Northern blot and semi-quantitative reverse transcription analyses showed that mouse VPS4a and VPS4b are differentially expressed in different organs, suggesting that the two paralogues have developed different functional properties since their divergence. To investigate the subcellular distribution of the murine VPS4 paralogues, we transiently expressed various fluorescent VPS4 fusion proteins in mouse 3T3 cells. All tested VPS4 fusion proteins were found in the cytosol. Expression of dominant-negative mutant VPS4 fusion proteins led to their concentration in the perinuclear region. Co-expression of VPS4a-GFP and VPS4b-dsRed fusion proteins revealed a partial co-localization that was most prominent with mutant VPS4a and VPS4b proteins. A physical interaction between the mouse paralogues was also supported by two-hybrid analyses.
Wiemann S, Weil B, Wellenreuther R, Gassenhuber J, Glassl S,
Ansorge W, Bocher M, Blocker H, Bauersachs S, Blum H, Lauber J,
Düsterhoft A, Beyer A, Köhrer K, Strack N, Mewes
HW, Ottenwälder B, Obermaier B, Tampe J, Heubner D,
Wambutt R, Korn B, Klein M, Poustka A.
Molecular Genome Analysis, German Cancer Research Center, 69120 Heidelberg, Germany. firstname.lastname@example.org
Genome Res. 2001 Mar;11(3):422-35.
With the complete human genomic sequence being unraveled, the focus will shift to gene identification and to the functional analysis of gene products. The generation of a set of cDNAs, both sequences and physical clones, which contains the complete and noninterrupted protein coding regions of all human genes will provide the indispensable tools for the systematic and comprehensive analysis of protein function to eventually understand the molecular basis of man. Here we report the sequencing and analysis of 500 novel human cDNAs containing the complete protein coding frame. Assignment to functional categories was possible for 52% (259) of the encoded proteins, the remaining fraction having no similarities with known proteins. By aligning the cDNA sequences with the sequences of the finished chromosomes 21 and 22 we identified a number of genes that either had been completely missed in the analysis of the genomic sequences or had been wrongly predicted. Three of these genes appear to be present in several copies. We conclude that full-length cDNA sequencing continues to be crucial also for the accurate identification of genes. The set of 500 novel cDNAs, and another 1000 full-coding cDNAs of known transcripts we have identified, adds up to cDNA representations covering 2% - 5% of all human genes. We thus substantially contribute to the generation of a gene catalog, consisting of both full-coding cDNA sequences and clones, which should be made freely available and will become an invaluable tool for detailed functional studies.
Wiemann S, Mehrle A, Bechtel S, Wellenreuther R,
Pepperkok R, Poustka A; German cDNA Consortium.
Molecular Genome Analysis, German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. email@example.com
C R Biol. 2003 Oct-Nov;326(10-11):1003-9.
To functionally characterize numerous novel proteins encoded by cDNAs sequenced by the German Consortium, 800 were tagged with green fluorescent protein. The subcellular localizations of the fusion proteins were examined in living cells, enabling their classification in subcellular groups. Their activity in cell growth, cell death, and protein transport was screened in high throughput using robotic liquid handling and reading stations. The resulting information is integrated with functional genomics and proteomics data for further understanding of protein functions in the cellular context.
Wiemann S, Bechtel S, Bannasch D, Pepperkok R, Poustka A;
German cDNA Network.
Molecular Genome Analysis, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany. firstname.lastname@example.org
J Struct Funct Genomics. 2003;4(2-3):87-96.
Among the greatest challenges facing biology today is the exploitation of huge amounts of genomic data, and their conversion into functional information about the proteins encoded. For example, the large-scale cDNA sequencing project of the German cDNA Consortium is providing vast numbers of open reading frames (ORFs) encoding novel proteins of completely unknown function. As a first step towards their characterization we have tagged over 500 of these with the green fluorescent protein (GFP), and examined the subcellular localizations of these fusion proteins in living cells. These data have allowed us to classify the proteins into subcellular groups which determines the next step towards a detailed functional characterization. To make further use of these GFP-tagged constructs, a series of functional assays have been designed and implemented to assess the effect of these novel proteins on processes such as cell growth, cell death, and protein transport. Functional assays with such a large set of molecules is only possible by automation. Therefore, we have developed, and adapted, functional assays for use by robotic liquid handling stations and reading stations. A transport assay allows to identify proteins which localize to distinct organelles of the secretory pathway and have the potential to be new regulators in protein transport, a proliferation assay helps identifying proteins that stimulate or repress mitosis. Further assays to monitor the effects of the proteins in apoptosis and signal transduction pathways are in progress. Integrating the functional information that is generated in the assays with data from expression profiling and further functional genomics and proteomics approaches, will ultimately allow us to identify functional networks of proteins in a morphological context, and will greatly contribute to our understanding of cell function.
Wellenreuther R, Schupp I, Poustka A, Wiemann S;
The German cDNA Consortium.
Department of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany. email@example.com.
BMC Genomics. 2004 Jun 15;5(1):36.
BACKGROUND: cDNA libraries are widely used to
identify genes and splice variants, and as a physical resource
for full-length clones. Conventionally-generated cDNA
libraries contain a high percentage of 5'-truncated clones.
Current library construction methods that enrich for
full-length mRNA are laborious, and involve several
enzymatic steps performed on mRNA, which renders them
sensitive to RNA degradation. The SMART technique for
full-length enrichment is robust but results in limited
cDNA insert size of the library.
RESULTS: We describe a method to construct SMART full-length enriched cDNA libraries with large insert sizes. Sub-libraries were generated from size-fractionated cDNA with an average insert size of up to seven kb. The percentage of full-length clones was calculated for different size ranges from BLAST results of over 12,000 5'ESTs.
CONCLUSIONS: The presented technique is suitable to generate full-length enriched cDNA libraries with large average insert sizes in a straightforward and robust way. The representation of full-coding clones is high also for large cDNAs (70%, 4-10 kb), when high-quality starting mRNA is used.
CA Confessio Augustana 3: S.45-52. Freimund-Verlag, 91561 Neuendettelsau (2001).
Im Aufsatz werden zunächst die Begriffe "Stammzellen", "Klonen" und "therapeutisches Klonen" sowie die biologisch-medizinischen Hintergründe erläutert. Ausgehend von der Frage, ob eine traditionelle, historisch gewachsene Ethik alleine überhaupt die nötige Trennschärfe aufweist, wird die Menschenwürde des Embryo diskutiert. Dabei argumentiere ich für eine gradualistische Sichtweise, in der der Embryo menschliche Eigenschaften und damit auch Anspruch auf Schutz langsam und Stück für Stück gewinnt. Davon ausgehend plädiere ich für eine Verantwortungsethik, die die Wertekonflikte sieht, akzeptiert und dann abwägt. Die dargelegte Sichtweise sieht deutlich den Wert des menschlichen Lebens an sich, unabhängig vom Alter und Entwicklungsstand. Dennoch kontrastiert sie stark gegen fundamentale (oder gar fundamentalistische) Ansichten, wonach der Mensch ab Zygote unantastbar sei.