Biomedical subjects

G H Braus

Publications and source records attributed to G H Braus.

At least 19 recordsLinked to original sources

PubMed2007

Cyclin-dependent kinase 5 is an upstream regulator of mitochondrial fission during neuronal apoptosis.

Under physiological conditions, mitochondrial morphology dynamically shifts between a punctuate appearance and tubular networks. However, little is known about upstream signal transduction pathways that regulate mitochondrial morphology. We show that mitochondrial fission is a very early and kinetically invariant event during neuronal cell death, which causally contributes to cytochrome c release and neuronal apoptosis. Using a small molecule CDK5 inhibitor, as well as a dominant-negative CDK5 mutant and RNAi knockdown experiments, we identified CDK5 as an upstream signalling kinase that regulates mitochondrial fission during apoptosis of neurons. Vice versa, our study shows that mitochondrial fission is a modulator contributing to CDK5-mediated neurotoxicity. Thereby, we provide a link that allows integration of CDK5 into established neuronal apoptosis pathways.

Animals↗

PubMed2005

Nitrogen metabolism of Aspergillus and its role in pathogenicity.

Aspergilli represent unique pathogens. Based on their saprophytic life style they are able to colonize a variety of ecological niches, among them the immunocompromised individual. Distinct fungal attributes that play a role in pathogenicity of aspergilli have been described, and primary metabolism indisputably has to be taken into account for contributing to the virulence potential of this fungal genus. Here we present an overview of studies that focus on this aspect of nutritional versatility. In the predominant pathogenic representative Aspergillus fumigatus regulation of nitrogen utilization and sensing of nitrogen sources have been scrutinized with respect to pathogenicity. The impact of distinct metabolic pathways on virulence capacities could be evaluated by inspection of auxotrophic mutant strains. Among them, para-aminobenzoic acid-requiring mutants revealed that this biosynthetic route is strictly required for pathogenicity. For amino acid anabolism only lysine biosynthesis has been investigated in this regard. Fungal amino acid biosynthesis is generally subject to strict regulation mediated by the Cross-Pathway Control system, a conserved regulatory circuit evolved to counteract conditions of nutritional stress. A clear influence of the system on pathogenicity could be observed by targeting its transcriptional activator CpcA. However, additional metabolic characteristics as well as regulatory instruments that compensate environmental challenges need to be addressed in future research with the aim to assess the significance of fungal primary metabolism for pathogenicity of aspergillus species.

Animals↗

PubMed2003

Nuclear import of yeast Gcn4p requires karyopherins Srp1p and Kap95p.

The yeast transcription factor Gcn4p contains two stretches of amino acid residues, NLS1 and NLS2, which are independently able to relocate the cytoplasmic protein chorismate mutase into the nucleus. Only NLS2 is conserved among fungi. A truncated version of CPCA (the counterpart of Gcn4p in Aspergillus nidulans), which lacks the conserved NLS, accumulates in the cytoplasm instead of the nucleus. Nuclear uptake mediated by the NLS1 of Gcn4p is impaired by defects in genes for several different karyopherins, whereas NLS2-dependent nuclear import specifically requires the alpha-importin Srp1p and the beta-importin Kap95p. Yeast strains that are defective in either of these two karyopherins are unable to respond to amino acid starvation. We have thus identified Gcn4p as a substrate for the Srp1p/Kap95p transport complex. Our data suggest that NLS2 is the essential and specific nuclear transport signal; NLS1 may play only an unspecific or accessory role.

Active Transport, Cell Nucleus↗

PubMed2003

Deletion of Aspergillus nidulans aroC using a novel blaster module that combines ET cloning and marker rescue.

Blaster cassettes are of significant value in functional genomics, as they represent tools with which to inactivate duplicated or homologous genes in an individual organism. We have constructed a novel blaster module which allows repeated gene deletion in the filamentous fungus Aspergillus nidulans. Because bacterial resistance marker cassettes are employed as flanking repeats in direct orientation, the blaster cassette is suited for recombinogenic engineering by ET cloning in Escherichia coli. The functionality of the blaster module was demonstrated by deleting the chorismate mutase-encoding gene aroC of A. nidulans, followed by marker rescue based on mitotic recombination. The resulting aroCDelta strains are auxotrophic for phenylalanine but not tyrosine, and display a limited capacity for fruit body formation and ascosporogenesis, which depends on the phenylalanine/tyrosine supply. The data support the notion that amino acid status has a strong impact on cleistothecium development in A. nidulans.

Aspergillus nidulans↗

PubMed2001

Repression of GCN4 mRNA translation by nitrogen starvation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae activates a regulatory network called "general control" that provides the cell with sufficient amounts of protein precursors during amino acid starvation. We investigated how starvation for nitrogen affects the general control regulatory system, because amino acid biosynthesis is part of nitrogen metabolism. Amino acid limitation results in the synthesis of the central transcription factor Gcn4p, which binds to specific DNA-binding motif sequences called Gcn4-protein-responsive elements (GCREs) that are present in the promoter regions of its target genes. Nitrogen starvation increases GCN4 transcription but efficiently represses expression of both a synthetic GCRE6::lacZ reporter gene and the natural amino acid biosynthetic gene ARO4. Repression of Gcn4p-regulated transcription by nitrogen starvation is independent of the ammonium sensing systems that include Mep2p and Gpa2p or Ure2p and Gln3p but depends on the four upstream open reading frames in the GCN4 mRNA leader sequence. Efficient translation of GCN4 mRNA is completely blocked by nitrogen starvation, even when cells are simultaneously starved for amino acids and eukaryotic initiation factor-2 alpha is fully phosphorylated by Gcn2p. Our data suggest that nitrogen starvation regulates translation of GCN4 by a novel mechanism that involves the four upstream open reading frames but that still acts independently of eukaryotic initiation factor-2 alpha phosphorylation by Gcn2p.

DNA-Binding Proteins↗

PubMed2001

Regulation of hisHF transcription of Aspergillus nidulans by adenine and amino acid limitation.

The hisHF gene of Aspergillus nidulans encodes imidazole-glycerole-phosphate (IGP) synthase, consisting of a glutamine amidotransferase and a cyclase domain. The enzyme catalyzes the fifth and sixth steps of histidine biosynthesis, which results in an intermediate of the amino acid and an additional intermediate of purine biosynthesis. An A. nidulans hisHF cDNA complemented a Saccharomyces cerevisiae his7Delta strain and Escherichia coli hisH and hisF mutant strains. The genomic DNA encoding the hisHF gene was cloned and its sequence revealed two introns within the 1659-bp-long open reading frame. The transcription of the hisHF gene of A. nidulans is activated upon amino acid starvation, suggesting that hisHF is a target gene of cross pathway control. Adenine but not histidine, both end products of the biosynthetic pathways connected by the IGP synthase, represses hisHF transcription. In contrast to other organisms HISHF overproduction did not result in any developmental phenotype of the fungus in hyphal growth or the asexual life cycle. hisHF overexpression caused a significantly reduced osmotic tolerance and the inability to undergo the sexual life cycle leading to acleistothecial colonies.

Adenine↗

PubMed2001

Regulative fine-tuning of the two novel DAHP isoenzymes aroFp and aroGp of the filamentous fungus Aspergillus nidulans.

Two novel genes, aroF and aroG, from the filamentous fungus Aspergillus nidulans were isolated and the regulative fine-tuning between the encoded, differentially regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthases was analyzed. A wide range of DAHP synthase isoenzymes of various organisms are known, but only a few have been characterized further. DAHP synthases (EC 4.1.2.15) catalyze the first committed step of the shikimate pathway, which is a putative target for anti-weed drugs. The reaction is the condensation of erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to yield DAHP. The two purified DAHP synthases showed different affinities for the substrates: 175 microM for PEP and 341 microM for E4P for the aroFp isoenzyme and weaker affinities of 239 microM (PEP) and 475 microM (E4P) for the aroGp isoenzyme. The enzymes are differentially regulated by tyrosine (aroFp) and phenylalanine (aroGp). The calculated kcat values are 7.0 s-1 for the tyrosine-inhibitable (aroFp) and 5.5 s-1 for the phenylalanine inhibitable (aroGp) enzyme. Tyrosine is a competitive inhibitor of the aroFp DAHP synthase in its reaction with PEP. Phenylalanine is a competitive inhibitor of the isoenzyme aroGp in its reaction with E4P. Both enzymes are inhibited by the chelating agent EDTA, which indicates a metal ion as cofactor.

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed2001

Regulation of the Aspergillus nidulans hisB gene by histidine starvation.

The hisB gene of the filamentous fungus Aspergillus nidulans encodes imidazole glycerol-phosphate dehydratase (E.C. 4.2.1.19), which catalyses the seventh enzymatic step in histidine biosynthesis. The gene was isolated and its deduced peptide sequence of 247 amino acids showed up to 54% identity with the IGPD enzymes of organisms comprising all three kingdoms. Expression of hisB cDNA in a Saccharomyces cerevisiae his3delta mutant strain functionally complemented the growth phenotype under histidine limitation. Addition of histidine did not affect hisB mRNA levels in A. nidulans wild-type cells. Histidine starvation conditions increased the hisB transcript level four-fold, suggesting regulation by a cross-pathway regulatory network. Deletion of the complete hisB open reading frame in A. nidulans strain A234 resulted in histidine auxotrophy. Additionally, hisB deletion strains were blocked from sexual fruiting body formation on medium containing low concentrations of histidine. This developmental phenotype of the hisB deletion mutant strain correlated with the induction of the cross-pathway control system.

Alcohol Oxidoreductases↗

PubMed2001

Induction of jlbA mRNA synthesis for a putative bZIP protein of Aspergillus nidulans by amino acid starvation.

The jlbA (jun-like hZIP) gene of Aspergillus nidulans was isolated. The deduced amino acid motif of the C-terminal region of jlhA encodes a putative DNA-binding site composed of a basic amino acid domain and an adjacent leucine zipper motif. This region shares highest similarities to the C-terminal DNA-binding domain and the basic zipper (bZIP)-motifs of transcription factors like CPCA from A. niger, Gcn4p from Saccharomyces cerevisiae, human JUNB and c-JUN. The putative jlbA protein contains a PEST-rich region (an instability region rich in the amino acids proline, glutamic acid, serine and threonine) described to be implicated in protein stability. The jlbA mRNA formation is elevated up to 40-fold upon amino acid starvation induced by the addition of the false feedback inhibitor 3-amino-1,2,4-triazole. This induction is partially dependent and partially independent on the presence of the transcription factor CPCA. Therefore jlbA is a novel gene of A. nidulans which is transcriptionally activated by amino acid starvation conditions.

Amino Acid Sequence↗

PubMed2001

Transcriptional autoregulation and inhibition of mRNA translation of amino acid regulator gene cpcA of filamentous fungus Aspergillus nidulans.

The CPCA protein of the filamentous fungus Aspergillus nidulans is a member of the c-Jun-like transcriptional activator family. It acts as central transcription factor of the cross-pathway regulatory network of amino acid biosynthesis and is functionally exchangeable for the general control transcriptional activator Gcn4p of Saccharomyces cerevisiae. In contrast to GCN4, expression of cpcA is strongly regulated by two equally important mechanisms with additive effects that lead to a fivefold increased CPCA protein amount under amino acid starvation conditions. One component of cpcA regulation involves a transcriptional autoregulatory mechanism via a CPCA recognition element (CPRE) in the cpcA promoter that causes a sevenfold increased cpcA mRNA level when cells are starved for amino acids. Point mutations in the CPRE cause a constitutively low mRNA level of cpcA and a halved protein level when amino acids are limited. Moreover, two upstream open reading frames (uORFs) in the 5' region of the cpcA mRNA are important for a translational regulatory mechanism. Destruction of both short uORFs results in a sixfold increased CPCA protein level under nonstarvation conditions and a 10-fold increase under starvation conditions. Mutations in both the CPRE and uORF regulatory elements lead to an intermediate effect, with a low cpcA mRNA level but a threefold increased CPCA protein level independent of amino acid availability. These data argue for a combined regulation of cpcA that includes a translational regulation like that of yeast GCN4 as well as a transcriptional regulation like that of the mammalian jun and fos genes.

Alleles↗

PubMed2001

Sexual diploids of Aspergillus nidulans do not form by random fusion of nuclei in the heterokaryon.

The sexual stage of Aspergillus (Emericella) nidulans consists of cleistothecia containing asci, each with eight ascospores. The fungus completes the sexual cycle in a homokaryotic or a heterokaryotic mycelium, respectively. The common assumption for the last 50 years was that different nuclear types are not distinguishable when sexual development is initiated. When cultured on a medium limited for glucose supplemented with 2% sorbitol, sexual development of A. nidulans is slowed and intact tetrads can be isolated. Through tetrad analysis we found that unlike haploid nuclei fuse preferentially to the prezygotic diploid nucleus. When heterokaryons are formed between nuclei of different genetic backgrounds, then recombinant asci derived from opposite nuclei are formed exclusively. Strains in the same heterokaryon compatibility group with moderate differences in their genetic backgrounds can discriminate between the nuclei of a heterokaryon and preferentially form a hybrid diploid nucleus, resulting in 85% recombinant tetrads. A. nidulans strains that differ at only a single genetic marker fuse the haploid nuclei at random for formation of diploid nuclei during meiosis. These results argue for a genetically determined "relative heterothallism" of nuclear recognition within a heterokaryon and a specific recruitment of different nuclei for karyogamy when available.

Aspergillus nidulans↗

PubMed2001

Different domains of the essential GTPase Cdc42p required for growth and development of Saccharomyces cerevisiae.

In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth. Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth. In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression. In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth. Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions. Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.

Actins↗

PubMed2001

Allosteric regulation of catalytic activity: Escherichia coli aspartate transcarbamoylase versus yeast chorismate mutase.

Allosteric regulation of key metabolic enzymes is a fascinating field to study the structure-function relationship of induced conformational changes of proteins. In this review we compare the principles of allosteric transitions of the complex classical model aspartate transcarbamoylase (ATCase) from Escherichia coli, consisting of 12 polypeptides, and the less complicated chorismate mutase derived from baker's yeast, which functions as a homodimer. Chorismate mutase presumably represents the minimal oligomerization state of a cooperative enzyme which still can be either activated or inhibited by different heterotropic effectors. Detailed knowledge of the number of possible quaternary states and a description of molecular triggers for conformational changes of model enzymes such as ATCase and chorismate mutase shed more and more light on allostery as an important regulatory mechanism of any living cell. The comparison of wild-type and engineered mutant enzymes reveals that current textbook models for regulation do not cover the entire picture needed to describe the function of these enzymes in detail.

Allosteric Regulation↗

PubMed2000

Asymmetrically localized Bud8p and Bud9p proteins control yeast cell polarity and development.

Diploid strains of the budding yeast Saccharomyces cerevisiae change the pattern of cell division from bipolar to unipolar when switching growth from the unicellular yeast form (YF) to filamentous, pseudohyphal (PH) cells in response to nitrogen starvation. The functions of two transmembrane proteins, Bud8p and Bud9p, in regulating YF and PH cell polarity were investigated. Bud8p is highly concentrated at the distal pole of both YF and PH cells, where it directs initiation of cell division. Asymmetric localization of Bud8p is independent of the Rsr1p/Bud1p GTPase. rsr1/bud1 mutations are epistatic to bud8 mutations, placing Rsr1p/Bud1p downstream of Bud8p. In YF cells, Bud9p is also localized at the distal pole, yet deletion of BUD9 favours distal bud initiation. In PH cells, nutritional starvation for nitrogen efficiently prevents distal localization of Bud9p. Because Bud8p and Bud9p proteins associate in vivo, we propose Bud8p as a landmark for bud initiation at the distal cell pole, where Bud9p acts as inhibitor. In response to nitrogen starvation, asymmetric localization of Bud9p is averted, favouring Bud8p-mediated cell division at the distal pole.

Cell Polarity↗

PubMed2000

Yeast Ran-binding protein Yrb1p is required for efficient proteolysis of cell cycle regulatory proteins Pds1p and Sic1p.

Ubiquitin-dependent proteolysis of specific target proteins is required for several important steps during the cell cycle. Degradation of such proteins is strictly cell cycle-regulated and triggered by two large ubiquitin ligases, termed anaphase-promoting complex (APC) and Skp1/Cullin/F-box complex (SCF). Here we show that yeast Ran-binding protein 1 (Yrb1p), a predominantly cytoplasmic protein implicated in nucleocytoplasmic transport, is required for cell cycle regulated protein degradation. Depletion of Yrb1p results in the accumulation of unbudded G(1) cells and of cells arrested in mitosis implying a function of Yrb1p in the G(1)/S transition and in the progression through mitosis. Temperature-sensitive yrb1-51 mutants are defective in APC-mediated degradation of the anaphase inhibitor protein Pds1p and in degradation of the cyclin-dependent kinase inhibitor Sic1p, a target of SCF. Thus, Yrb1p is crucial for efficient APC- and SCF-mediated proteolysis of important cell cycle regulatory proteins. We have identified the UBS1 gene as a multicopy suppressor of yrb1-51 mutants. Ubs1p is a nuclear protein, and its deletion is synthetic lethal with a yrb1-51 mutation. Interestingly, UBS1 was previously identified as a multicopy suppressor of cdc34-2 mutants, which are defective in SCF activity. We suggest that Ubs1p may represent a link between nucleocytoplasmic transport and ubiquitin ligase activity.

Anaphase↗

PubMed2000

Coevolution of transcriptional and allosteric regulation at the chorismate metabolic branch point of Saccharomyces cerevisiae.

Control of transcription and enzyme activities are two interwoven regulatory systems essential for the function of a metabolic node. Saccharomyces cerevisiae strains differing in enzyme activities at the chorismate branch point of aromatic amino acid biosynthesis were constructed by recombinant DNA technology. Expression of an allosterically unregulated, constitutively activated chorismate mutase encoded by the ARO7(T226I) (ARO7(c)) allele depleted the chorismate pool. The resulting tryptophan limitation caused growth defects, which could be counteracted only by transcriptional induction of TRP2 encoding the competing enzyme anthranilate synthase. ARO7 expression is not transcriptionally regulated by amino acids. Transcriptional activation of the ARO7(c) allele led to stronger growth retardation upon tryptophan limitation. The same effect was achieved by removing the competing enzyme anthranilate synthase, which is encoded by the TRP2 gene, from the transcriptional control. The allelic situation of ARO7(c) being under general control instead of TRP2 resulted in severe growth defects when cells were starved for tryptophan. In conclusion, the specific regulatory pattern acting on enzymatic activities at the first metabolic node of aromatic amino acid biosynthesis is necessary to maintain proper flux distribution. Therefore, the evolution of the sophisticated allosteric regulation of yeast chorismate mutase requires as prerequisite (i) that the encoding ARO7 gene is not transcriptionally regulated, whereas (ii) the transcription of the competing feedback-regulated anthranilate synthase-encoding gene is controlled by availability of amino acids.

Alleles↗

PubMed2000

Two different modes of cyclin clb2 proteolysis during mitosis in Saccharomyces cerevisiae.

Sister chromatid separation and mitotic exit are triggered by the anaphase-promoting complex (APC/C) which is a multi-subunit ubiquitin ligase required for proteolytic degradation of various target proteins. Cdc20 and Cdh1 are substrate-specific activators of the APC/C. It was previously proposed that Cdh1 is essential for proteolysis of the yeast mitotic cyclin Clb2. We show that Clb2 proteolysis is triggered by two different modes during mitosis. A fraction of Clb2 is degraded during anaphase in the absence of Cdh1. However, a second fraction of Clb2 remains stable during anaphase and is degraded in a Cdh1-dependent manner as cells exit from mitosis. Most of cyclin Clb3 is degraded independently of Cdh1. Our data imply that degradation of mitotic cyclins is initiated by a Cdh1-independent mechanism.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2000

Developmental and metabolic regulation of the phosphoglucomutase-encoding gene, pgmB, of Aspergillus nidulans.

We have isolated the pgmB gene from Aspergillus nidulans, which encodes a phosphoglucomutase, one of the key enzymes in carbohydrate metabolism. The pgmB gene is located on chromosome VII and its ORF encodes 557 amino acids. Mutant phenotypes were analysed by expression of high levels of pgmB antisense RNA, which lead to a loss of detectable levels of sense RNA. Under conditions of antisense RNA expression, a 30% reduction in the growth rate was observed in comparison to wild-type. On the enzyme level, expression of pgmB antisense RNA resulted in a 35% reduction in total phosphoglucomutase activity. Two pgmB mRNAs were observed under all conditions tested and differ with respect to the location of the poly(A) site. Expression of pgmB driven by the GAL1 promoter in Saccharomyces cerevisiae complemented the growth phenotype of a pgm2delta mutant strain and suppressed the sensitivity of a gcn4delta mutant strain to amino acid starvation in the presence of galactose. Cultivation of A. nidulans in the presence of glucose or galactose as carbon source did not affect transcription of pgmB. However, amino acid starvation conditions resulted in a six-fold reduction in the level of pgmB mRNA, while genes for amino acid biosynthesis showed increased transcription. Transcription of pgmB was low during hyphal growth and in the sexual phase of development, but was significantly increased during the asexual stage of the A. nidulans life cycle.

Amino Acid Sequence↗