Biomedical subjects

F Lottspeich

Publications and source records attributed to F Lottspeich.

At least 19 recordsLinked to original sources

PubMed2005

Identification of Dll1 (Delta1) target genes during mouse embryogenesis using differential expression profiling.

The Notch signaling pathway has pleiotropic functions during mammalian embryogenesis. It is required for the patterning and differentiation of the presomitic and somitic paraxial mesoderm and of the neural tube. We used DNA-chip expression profiling and 2D-gel electrophoresis combined with peptide mass fingerprinting to identify genes and proteins differentially regulated in E10.5 Dll1 (delta-like 1, Delta1) mutant embryos. The differential expression profiling approach identified 47 regulated transcripts and 40 differentially expressed proteins. The majority of these genes has until now not been associated with Notch signaling. Subsequent whole-mount in situ hybridization confirmed that a subset of the identified transcripts has restricted and distinct patterns of expression in E10.5 mouse embryos. For most genes these expression patterns were affected in the presomitic mesoderm, in differentiating somites of Dll1 mutant embryos and in the neural tube and cells differentiating from it. Similar effects were observed in embryos homozygous for the Headturner (Htu) and pudgy (pu) mutations, which are alleles of the Notch ligands Jag1 and Dll3. The regulated expression of a subset of the proteins was validated by immunoblots. Remarkably six of the proteins down-regulated in Dll1 mutant embryos are proteasome subunits. The large set of regulated genes identified in this differential expression profiling approach is an important resource for further functional studies.

Animals↗

PubMed2005

Identification of coexpressed gene clusters in a comparative analysis of transcriptome and proteome in mouse tissues.

A major advantage of the mouse model lies in the increasing information on its genome, transcriptome, and proteome, as well as in the availability of a fast growing number of targeted and induced mutant alleles. However, data from comparative transcriptome and proteome analyses in this model organism are very limited. We use DNA chip-based RNA expression profiling and 2D gel electrophoresis, combined with peptide mass fingerprinting of liver and kidney, to explore the feasibility of such comprehensive gene expression analyses. Although protein analyses mostly identify known metabolic enzymes and structural proteins, transcriptome analyses reveal the differential expression of functionally diverse and not yet described genes. The comparative analysis suggests correlation between transcriptional and translational expression for the majority of genes. Significant exceptions from this correlation confirm the complementarities of both approaches. Based on RNA expression data from the 200 most differentially expressed genes, we identify chromosomal colocalization of known, as well as not yet described, gene clusters. The determination of 29 such clusters may suggest that coexpression of colocalizing genes is probably rather common.

Animals↗

PubMed2005

Identification and characterization of Cor33p, a novel protein implicated in tolerance towards oxidative stress in Candida albicans.

We applied two-dimensional gel electrophoresis to identify downstream effectors of CPH1 and EFG1 under hypha-inducing conditions in Candida albicans. Among the proteins that were expressed in wild-type cells but were strongly downregulated in a cph1Delta/efg1Delta double mutant in alpha-minimal essential medium at 37 degrees C, we could identify not-yet-characterized proteins, including Cor33-1p and Cor33-2p. The two proteins are almost identical (97% identity) and represent products of allelic isoforms of the same gene. Cor33p is highly similar to Cip1p from Candida sp. but lacks any significant homology to proteins from Saccharomyces cerevisiae. Strikingly, both proteins share homology with phenylcoumaran benzylic ether reductases and isoflavone reductases from plants. For other hypha-inducing media, like yeast-peptone-dextrose (YPD) plus serum at 37 degrees C, we could not detect any transcription for COR33 in wild-type cells, indicating that Cor33p is not hypha specific. In contrast, we found a strong induction for COR33 when cells were treated with 5 mM hydrogen peroxide. However, under oxidative conditions, transcription of COR33 was not dependent on EFG1, indicating that other regulatory factors are involved. In fact, upregulation depends on CAP1 at least, as transcript levels were clearly reduced in a Deltacap1 mutant strain under oxidative conditions. Unlike in wild-type cells, transcription of COR33 in a tsa1Delta mutant can be induced by treatment with 0.1 mM hydrogen peroxide. This suggests a functional link between COR33 and thiol-specific antioxidant-like proteins that are important in the oxidative-stress response in yeasts. Concordantly, cor33Delta deletion mutants show retarded growth on YPD plates supplemented with hydrogen peroxide, indicating that COR33 in general is implicated in conferring tolerance toward oxidative stress on Candida albicans.

Alleles↗

PubMed2004

Induction of apoptosis by a hepatocyte conditioned medium.

Incubation of primary cultures of parenchymal hepatocytes in a conditioned medium (CM), collected over the first 3 h of serum-free rat hepatocyte culture (CM(0-3)), induces a time dependent increase of the frequency of apoptotic cells which is accompanied by prominent changes of cell morphology. Short-term treatment with CM(0-3) for the first 3 h of culture is sufficient to significantly (P < 0.05) increase the frequency of apoptotic cells, however, the effect is more pronounced upon long-term treatment. Although apoptosis induction by CM(0-3) is independent of the timepoint when cultivation in CM(0-3) starts, our results suggest that the sensitivity for apoptosis induction by CM(0-3) is increased during the phase of attachment. Purification of CM(0-3) resulted in a fraction which significantly (P < 0.05) induced apoptosis at concentrations >/=10 ng/ml. Exposure of cultures to concentrations >/=1 microg/ml of purified CM(0-3) gave rise to a prominent cytotoxic effect as indicated by the massive occurrence of necrotic cells. Biochemical analysis showed that the purified fraction of CM(0-3) contains acidic ferritins with molecular weight of 23 and 43 kDa. Strikingly, both share homologies with placental isoferritins (PLF), for which growth inhibitory and immunosuppressive effects have been demonstrated by several investigations. Therefore, our results provide evidence that rat hepatocytes produce PLF or PLF-related acidic isoferritins which are able to induce apoptosis.

Animals↗

PubMed2003

A novel docking site on Mediator is critical for activation by VP16 in mammalian cells.

ARC92/ACID1 was identified as a novel specific target of the herpes simplex transactivator VP16 using an affinity purification procedure. Characterization of the protein revealed tight interactions with human Mediator mediated through a von Willebrand type A domain. ARC92/ACID1 further contains a novel activator-interacting domain (ACID), which it shares with at least one other human gene termed PTOV1/ACID2. The structure of ARC92/ACID1 is of ancient origin but is conserved in mammals and in selected higher eukaryotes. A subpopulation of Mediator is associated with ARC92/ACID1, which is specifically required for VP16 activation both in vitro and in mammalian cells, but is dispensable for other activators such as SP1. Despite many known targets of VP16, ARC92/ACID1 appears to impose a critical control on transcription activation by VP16 in mammalian cells.

Animals↗

PubMed2003

Limited proteolysis of surfactant protein D causes a loss of its calcium-dependent lectin functions.

Surfactant protein D (SP-D) is a multimeric collagenous lectin that mediates the clearance of pathogens and modulates immune cell functions via its C-terminal carbohydrate recognition domain (CRD). We hypothesized that extracellular proteolysis of SP-D may result in a loss of its functional properties. Multimeric SP-D was partially digested by human leukocyte elastase (HLE) dose- and time-dependently. Physiologic concentrations of calcium slowed, but did not protect from degradation. In solution, both native and degraded SP-D had an apparent molecular weight of 650 to >1000 kDa. Under reducing conditions, the degraded SP-D monomers run at 10 kDa less than native SP-D. Amino acid sequencing located all major cleavage sites into the CRD. Functional studies showed that degraded SP-D had lost its calcium-dependent lectin properties, i.e. neither bound to mannose nor agglutinated bacteria. These studies demonstrate that elastase results in the limited proteolysis of SP-D with loss of its CRD-dependent activities and suggest that proteases at concentrations observed in various lung diseases may impair the antimicrobial and immunomodulatory roles of SP-D.

Agglutination Tests↗

PubMed2003

Identification of cell surface determinants in Candida albicans reveals Tsa1p, a protein differentially localized in the cell.

To identify cell surface proteins of Candida albicans, the predominant fungal pathogen in humans, we have established an approach using a membrane impermeable biotin derivative in combination with affinity purification. We were able to identify 29 different proteins under two distinct conditions. Among mannoproteins, heat shock proteins and glycolytic enzymes we found thiol-specific antioxidant-like protein 1 (Tsa1p) to be differentially localized depending on the conditions applied. Only in hyphally grown cells Tsa1p was localized to the cell surface whereas in blastospores no surface but mainly nuclear localization was found. This indicates that cell surface expression of at least some proteins is mediated by differential translocation.

Biotinylation↗

PubMed2002

A gene that encodes a product with similarity to dioxygenases is highly expressed in teliospores of Ustilago maydis.

The phytopathogenic basidiomycete Ustilago maydis produces sexual teliospores only after infection of its host plant, maize. To investigate the process of spore formation, we have isolated Ssp1, a protein that is abundantly expressed in mature teliospores. The corresponding gene, ssp1, is expressed at low levels in haploid sporidia; however, transcriptional levels are drastically induced in mature teliospores. Transcriptional regulation of ssp1 involves positive and negative promoter elements, and is subject to control by the cAMP signaling cascade and histone deacetylase-mediated repression. Ssp1 shows similarity to linoleate diol synthase, a fatty acid dioxygenase from the fungus Gaeumannomyces graminis. In agreement with this presumed function, Ssp1 is localized on lipid bodies in germinating teliospores, suggesting a role in the mobilization of storage lipids.

Amino Acid Sequence↗

PubMed2001

Recruitment to Golgi membranes of ADP-ribosylation factor 1 is mediated by the cytoplasmic domain of p23.

Binding to Golgi membranes of ADP ribosylation factor 1 (ARF1) is the first event in the initiation of COPI coat assembly. Based on binding studies, a proteinaceous receptor has been proposed to be critical for this process. We now report that p23, a member of the p24 family of Golgi-resident transmembrane proteins, is involved in ARF1 binding to membranes. Using a cross-link approach based on a photolabile peptide corresponding to the cytoplasmic domain of p23, the GDP form of ARF1 (ARF1-GDP) is shown to interact with p23 whereas ARF1-GTP has no detectable affinity to p23. The p23 binding is shown to localize specifically to a 22 amino acid C-terminal fragment of ARF1. While a monomeric form of a non-photolabile p23 peptide does not significantly inhibit formation of the cross-link product, the corresponding dimeric form does compete efficiently for this interaction. Consistently, the dimeric p23 peptide strongly inhibits ARF1 binding to native Golgi membranes suggesting that an oligomeric form of p23 acts as a receptor for ARF1 before nucleotide exchange takes place.

ADP-Ribosylation Factor 1↗

PubMed2001

Molecular cloning and expression of human bile acid beta-glucosidase.

A novel microsomal beta-glucosidase was recently purified and characterized from human liver that catalyzes the hydrolysis of bile acid 3-O-glucosides as endogenous compounds. The primary structure of this bile acid beta-glucosidase was deduced by cDNA cloning on the basis of the amino acid sequences of peptides obtained from the purified enzyme by proteinase digestion. The isolated cDNA comprises 3639 base pairs containing 524 nucleotides of 5'-untranslated and 334 nucleotides of 3'-untranslated sequences including the poly(A) tail. The open reading frame predicts a 927-amino acid protein with a calculated M(r) of 104,648 containing one putative transmembrane domain. Data base searches revealed no homology with any known glycosyl hydrolase or other functionally identified protein. The cDNA sequence was found with significant identity in the human chromosome 9 clone RP11-112J3 of the human genome project. The recombinant enzyme was expressed in a tagged form in COS-7 cells where it displayed bile acid beta-glucosidase activity. Northern blot analysis of various human tissues revealed high levels of expression of the bile acid beta-glucosidase mRNA (3.6-kilobase message) in brain, heart, skeletal muscle, kidney, and placenta and lower levels of expression in the liver and other organs.

Amino Acid Sequence↗

PubMed2001

Recruitment of cortexillin into the cleavage furrow is controlled by Rac1 and IQGAP-related proteins.

Cytokinesis in eukaryotic organisms is under the control of small GTP-binding proteins, although the underlying molecular mechanisms are not fully understood. Cortexillins are actin-binding proteins whose activity is crucial for cytokinesis in Dictyostelium. Here we show that the IQGAP-related and Rac1-binding protein DGAP1 specifically interacts with the C-terminal, actin-bundling domain of cortexillin I. Like cortexillin I, DGAP1 is enriched in the cortex of interphase cells and translocates to the cleavage furrow during cytokinesis. The activated form of the small GTPase Rac1A recruits DGAP1 into a quaternary complex with cortexillin I and II. In DGAP1(-) mutants, a complex can still be formed with a second IQGAP-related protein, GAPA. The simultaneous elimination of DGAP1 and GAPA, however, prevents complex formation and localization of the cortexillins to the cleavage furrow. This leads to a severe defect in cytokinesis, which is similar to that found in cortexillin I/II double-null mutants. Our observations define a novel and functionally significant signaling pathway that is required for cytokinesis.

Amino Acid Sequence↗

PubMed2001

Bioanalytical characterization of proteins.

Allergens from the view of a protein chemist are quite normal proteins, not to distinguish from non allergenic proteins. The first task is therefore to recognize and identify the proteins responsible for the allergenic reaction. This is usually only possible if the allergenic structure is conserved during the purification procedures. For a detailed analysis of the allergenic protein modern protein chemical methods for characterization, identification, determination of posttranslational modifications and epitope characterization have to be applied. Such techniques are briefly described in this article.

Allergens↗

PubMed2001

The ricinosomes of senescing plant tissue bud from the endoplasmic reticulum.

The ricinosome (synonym, precursor protease vesicle) is a novel organelle, found so far exclusively in plant cells. Electron microscopic studies suggest that it buds off from the endoplasmic reticulum in senescing tissues. Biochemical support for this unusual origin now comes from the composition of the purified organelle, which contains large amounts of a 45-kDa cysteine endoprotease precursor with a C-terminal KDEL motif and the endoplasmic reticulum lumen residents BiP (binding protein) and protein disulfide isomerase. Western blot analysis, peptide sequencing, and mass spectrometry demonstrate retention of KDEL in the protease proform. Acidification of isolated ricinosomes causes castor bean cysteine endopeptidase activation, with cleavage of the N-terminal propeptide and the C-terminal KDEL motif. We propose that ricinosomes accumulate during senescence by programmed cell death and are activated by release of protons from acidic vacuoles.

Amino Acid Sequence↗

PubMed2001

The hydH/G Genes from Escherichia coli code for a zinc and lead responsive two-component regulatory system.

The hydH/G genes from Escherichia coli code for a two-component regulatory system that has been implicated in the regulation of hydrogenase 3 formation. In a detailed study of the function of HydH/G employing hycA'-'lacZ reporter gene fusions, it was shown that HydH/G indeed led to a stimulation of activation of the hycA promoter responsible for hydrogenase 3 synthesis but only when hydG is overexpressed from a plasmid in a strain lacking FhlA. Since the stimulation was not observed with an fdhF'-'lacZ fusion, and since it was independent from a functional hydH gene product, it must be considered as unspecific cross-talk. An extensive search for the actual physiological signal of HydH/G showed that the system responds to high concentrations of zinc or lead in the medium. Expression of zraP, a gene inversely oriented to hydH/G whose product seems to be involved in acquisition of tolerance to high Zn(2+) concentrations, is stimulated by high Zn(2+) and Pb(2+) concentrations and this stimulation requires both HydH and HydG. Purified HydG in the presence of phosphoryl donors binds to a region within the zraP-hydHG intergenic region that is characterised by two inverted repeats separated by a 14 bp spacer. Putative -12/-24 sigma(54)-dependent promoter motifs are present upstream of both the zraP and the hydHG transcriptional units; in accordance, transcription of zraP is strictly dependent on the presence of a functional rpoN gene. The expression of hydH/G is autoregulated: high Zn(2+) and Pb(2+) concentrations lead to a significant increase of the HydG protein content which took place only in a hydH(+) genetic background. Since HydH binds to membranes tightly, it is assumed that the HydH/G system senses high periplasmic Zn(2+) and Pb(2+) concentrations and contributes to metal tolerance by activating the expression of zraP. The redesignation of hydH/G as zraS/R is suggested.

Bacterial Proteins↗

PubMed2001

Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin.

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).

Actins↗

PubMed2001

A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment.

We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Identification and characterization of neutral endopeptidase (EC 3. 4. 24. 11) from human prostasomes--localization in prostatic tissue and cell lines.

BACKGROUND: An antibody directed against a 100 kDa protein was immunoselected from a polyvalent antiserum against human prostasomes. The antibody as well as biochemical characteristics of the respective antigen were used to study the structural relationship of the latter with prostate membrane specific antigen (PMSA), another 100 kDa membrane protein of the prostate. METHODS: The isolated purified 100 kDa protein was characterized by tryptic degradation, aminoacid-sequencing and mass spectroscopy peptide-fingerprinting as well as mono-saccharide analysis and lectin binding and identified as a prostasomal neutral endopeptidase (NEP, EC 3.4.24.11). Immunohistochemistry, immunoelectron microscopy, in situ hybridization, and RT-PCR were performed to analyze the expression and distribution of the protein in normal and malignant human prostatic tissues and cell lines. RESULTS: Prostatic NEP, which has no relationship with PMSA, is a glycosylated, integral membrane protein type II. The prevalent glycosyl residues are NeuNAc, GlcNAc, GalNAc, Gal, Man, Fuc. NEP-mRNA is expressed in human prostatic epithelial and some stromal cells. NEP-immunoreactivity is strong in normal prostatic epithelium and confined to the apical plasma membrane. During apocrine secretion, the enzyme is released from the secretory cells, contributing to the formation of prostasomes. In prostate cancer specimens, immunoreactivity of apical plasma membranes is lost, while generalized cytoplasmic immunoreactivity develops. CONCLUSIONS: Prostatic secretory cells contain a membrane-bound, highly glycosylated neutral endopeptidase which is restricted to the apical plasma membrane. The enzyme is released from the cells in an apocrine fashion and contributes to the formation of prostasomes. In prostate cancer cells a preferential cytoplasmic localization is observed, pointing to alterations in intracellular targeting.

Amino Acid Sequence↗

PubMed2001

Identification of nucleolin as a glucocorticoid receptor interacting protein.

The glucocorticoid receptor (GR) is a ligand-induced transcription factor which modulates the transcriptional activity of target genes. Full transcriptional activity of GR is achieved with the help of accessory proteins that are able to interact with GR. We have identified a 95-kDa protein by a blotting technique which utilizes a radioactively labeled DNA-bound GR to detect proteins that bind to this complex. Biochemical purification of this protein followed by protein microsequencing resulted in the identification of human nucleolin. In addition we could show that a GR-deletion mutant localizes to the nucleolus, where nucleolin is one of the most abundant proteins. The binding of nucleolin to this deletion mutant was demonstrated by GST-pull-down experiments. We suggest a biological role of nucleolin in binding of GR in the nucleolus.

Animals↗