Biomedical subjects

Friedrich Lottspeich

Publications and source records attributed to Friedrich Lottspeich.

At least 19 recordsLinked to original sources

PubMed2006

Tyrosine phosphorylated Par3 regulates epithelial tight junction assembly promoted by EGFR signaling.

The conserved polarity complex, comprising the partitioning-defective (Par) proteins Par3 and Par6, and the atypical protein kinase C, functions in various cell-polarization events and asymmetric cell divisions. However, little is known about whether and how external stimuli-induced signals may regulate Par3 function in epithelial cell polarity. Here, we found that Par3 was tyrosine phosphorylated through phosphoproteomic profiling of pervanadate-induced phosphotyrosine proteins. We also demonstrated that the tyrosine phosphorylation event induced by multiple growth factors including epidermal growth factor (EGF) was dependent on activation of Src family kinase (SFK) members c-Src and c-Yes. The tyrosine residue 1127 (Y1127) of Par3 was identified as the major EGF-induced phosphorylation site. Moreover, we found that Y1127 phosphorylation reduced the association of Par3 with LIM kinase 2 (LIMK2), thus enabling LIMK2 to regulate cofilin phosphorylation dynamics. Substitution of Y1127 for phenylalanine impaired the EGF-induced Par3 and LIMK2 dissociation and delayed epithelial tight junction (TJ) assembly considerably. Collectively, these data suggest a novel, phosphotyrosine-dependent fine-tuning mechanism of Par3 in epithelial TJ assembly controlled by the EGF receptor-SFK signaling pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Temperature-sensitive eIF5A mutant accumulates transcripts targeted to the nonsense-mediated decay pathway.

The highly conserved protein eIF5A found in Archaea and all eukaryotes uniquely contains the posttranslationally formed amino acid hypusine. Despite being essential the functions of this protein and its modification remain unclear. To gain more insight into these functions temperature-sensitive mutants of the human EIF5A1 were characterized in the yeast Saccharomyces cerevisiae. Expression of the point mutated form V81G in a DeltaeIF5A strain of yeast led to a strongly temperature-sensitive phenotype and to a significantly reduced protein level at restrictive temperature. The mutant showed accumulation of a subset of mRNAs that was also observed in nonsense-mediated decay (NMD)-deficient yeast strains. After short incubation at restrictive temperature the mutant exhibited increased half-lives of the intron containing CYH2 pre-mRNA and mature transcripts of NMD-dependent genes. Reduced telomere silencing and shortening was detected in the V81G mutant further supporting similarities to NMD-deficient strains. Our data suggest that eIF5A mediates important cellular processes like cell viability and senescence through its effects on the stability of certain mRNAs.

Humans↗

PubMed2006

Quantitative profiling of the membrane proteome in a halophilic archaeon.

We present a large scale quantitation study of the membrane proteome from Halobacterium salinarum. To overcome problems generally encountered with membrane proteins, we established a membrane preparation protocol that allows the application of most proteomic techniques originally developed for soluble proteins. Proteins were quantified using two complementary approaches. For gel-based quantitation, DIGE labeling was combined with two-dimensional gel electrophoresis on an improved 16-benzyldimethyl-n-hexadecylammonium chloride/SDS system. MS-based quantitation was carried out by combining gel-free separation with the recently developed isotope-coded protein labeling technique. Good correlations between these two independent quantitation strategies were obtained. From computational analysis we conclude that labeling of free amino groups by isotope-coded protein labeling (Lys and free N termini) is better suited for membrane proteins than Cys-based labeling strategies but that quantitation of integral membrane proteins remains cumbersome compared with soluble proteins. Nevertheless we could quantify 155 membrane proteins; 101 of these had transmembrane domains. We compared two growth states that strongly affect the energy supply of the cells: aerobic versus anaerobic/phototrophic conditions. The photosynthetic protein bacteriorhodopsin is the most highly regulated protein. As expected, several other membrane proteins involved in aerobic or anaerobic energy metabolism were found to be regulated, but in total, however, the number of regulated proteins is rather small.

Cell Membrane↗

PubMed2006

Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences.

Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors.

Amino Acid Sequence↗

PubMed2006

Quantitative analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome alterations in 5L rat hepatoma cells using isotope-coded protein labels.

In an effort to contribute to a better understanding of the hepatic toxicity of the ubiquitous environmental pollutant and hepatocarcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a comprehensive quantitative proteome analysis was performed on 5L rat hepatoma cells exposed to 1 nM TCDD for 8 h. Changes in the abundances of individual protein species in TCDD-treated cells as compared to untreated cells were analysed using the nongel-based isotope-coded protein label (ICPL) method [Schmidt, A., Kellermann, J., Lottspeich, F., Proteomics 2005, 5, 4-15]. 89 proteins were identified as up- or down-regulated by TCDD. For the majority of the altered proteins, an impact of TCDD on their abundance had not been known before. Due to the physicochemical properties or the translational regulation of a large number of the affected proteins, their alteration would have escaped detection by gel-based methods for proteome analysis and by standard mRNA expression profiling, respectively. The identified proteins with TCDD-altered abundance include several proteins implicated in cell cycle regulation, growth factor signalling and the control of apoptosis. The results thus provide new starting-points for the investigation of specific aspects of the toxicity and carcinogenicity of dioxin in liver.

Animals↗

PubMed2006

Hypophosphorylation of the architectural chromatin protein DEK in death-receptor-induced apoptosis revealed by the isotope coded protein label proteomic platform.

During apoptosis nuclear morphology changes dramatically due to alterations of chromatin architecture and cleavage of structural nuclear proteins. To characterize early events in apoptotic nuclear dismantling we have performed a proteomic study of apoptotic nuclei. To this end we have combined a cell-free apoptosis system with a proteomic platform based on the differential isotopic labeling of primary amines with N-nicotinoyloxy-succinimide. We exploited the ability of this system to produce nuclei arrested at different stages of apoptosis to analyze proteome alterations which occur prior to or at a low level of caspase activation. We show that the majority of proteins affected at the onset of apoptosis are involved in chromatin architecture and RNA metabolism. Among them is DEK, an architectural chromatin protein which is linked to autoimmune disorders. The proteomic analysis points to the occurrence of multiple PTMs in early apoptotic nuclei. This is confirmed by showing that the level of phosphorylation of DEK is decreased following apoptosis induction. These results suggest the unexpected existence of an early crosstalk between cytoplasm and nucleus during apoptosis. They further establish a previously unrecognized link between DEK and cell death, which will prove useful in the elucidation of the physiological function of this protein.

Animals↗

PubMed2006

Proteome alterations in human host cells infected with coxsackievirus B3.

Coxsackievirus B3 (CVB3) is a common factor in human myocarditis. The interplay between host factors and virus components is crucial for the fate of the infected cells. Despite that, host protein responses, which characterize CVB3-induced diseases, have not yet been determined in detail. To investigate the nature of modified protein patterns in infected human cells compared with uninfected cells, two-dimensional gel electrophoresis in combination with matrix-assisted laser desorption/ionization-mass spectrometry were used. The regulated proteins, e.g. nucleophosmin (nucleolar protein B23), lamin, the RNA-binding protein UNR and the p38 mitogen-activated protein kinase, were sorted according to their functional groups and interpreted in the context of the myocarditis process.

Cell Line↗

PubMed2005

The highly abundant protein Ag-lbp55 from Ascaridia galli represents a novel type of lipid-binding proteins.

Lipid-binding proteins exhibit important functions in lipid transport, cellular signaling, gene transcription, and cytoprotection. Their functional analogues in nematodes are nematode polyprotein allergens/antigens and fatty acid and retinoid-binding proteins. This work describes a novel 55-kDa protein, Ag-lbp55, purified from the parasitic nematode Ascaridia galli. By direct N-terminal sequencing, a partial amino acid sequence was obtained that allowed the design of oligonucleotide primers to obtain the full-length cDNA sequence. Sequence analysis revealed the presence of an N-terminal signal peptide of 25 amino acid residues and a FAR domain at the C terminus. Data base searches showed almost no significant homologies to other described proteins. The secondary structure of Ag-lbp55 was predominantly alpha-helical (65%) as shown by CD spectroscopy. It was found to bind with high affinity fatty acids (caprylic, oleic, and palmitic acid) and their fluorescent analogue dansylaminoundecanic acid. Immunolocalization showed that Ag-lbp55 is a highly abundant protein, mainly distributed in the inner hypodermis and extracellularly in the pseudocoelomatic fluid. A similar staining pattern was observed in other pathogenic nematodes, indicating the existence of similar proteins in these species.

Amino Acid Sequence↗

PubMed2005

Identification and characterization of PorH, a new cell wall channel of Corynebacterium glutamicum.

The cell wall of Corynebacterium glutamicum contains the cation-selective channel (porin) PorA(C.glut) and the anion-selective channel PorB(C.glut) for the passage of hydrophilic solutes. Lipid bilayer experiments with organic solvent extracts of whole C. glutamicum cells cultivated in minimal medium suggested that also another cation-selective channel-forming protein, named PorH(C.glut), is present in C. glutamicum. The protein was purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column. The pure protein had an apparent molecular mass of about 12 kDa on SDS-PAGE. Western blot analysis suggested that the cell wall channel is presumably formed by protein oligomers. The purified protein forms cation-selective channels with an average single-channel conductance of about 2.5 nS in 1 M KCl in the lipid bilayer assay. The PorH(C.glut) protein was partially sequenced, and based on the resulting amino acid sequence, the corresponding gene, designated as porH(C.glut), was identified in the published genome sequence of C. glutamicum ATCC13032. PorH(C.glut) contains only the inducer methionine but no N-terminal extension, which suggests that the export and assembly of the protein follow a yet unknown pathway. PorH(C.glut) is coded in the bacterial chromosome by a gene that is localized in the vicinity of porA(C.glut), within a putative operon of 13 genes. RT-PCR revealed that both porins are cotranscribed. They coexist according to immunological detection experiments in the cell wall of C. glutamicum together with PorB(C.glut) and PorC(C.glut).

Amino Acid Sequence↗

PubMed2005

B7/CD28 costimulation of T cells induces a distinct proteome pattern.

Effective immune strategies for the eradication of human tumors require a detailed understanding of the interaction of tumor cells with the immune system, which might lead to an optimization of T cell responses. To understand the impact of B7-mediated costimulation on T cell activation comprehensive proteome analysis of B7-primed T cell populations were performed. Using this approach we identified different classes of proteins in T cells whose expression is either elevated or reduced upon B7-1- or B7-2-mediated CD28 costimulation. The altered proteins include regulators of the cell cycle and cell proliferation, signal transducers, components of the antigen processing machinery, transporters, cytoskeletal proteins, and metabolic enzymes. A number of differentially expressed proteins are further modified by phosphorylation. Our results provide novel insights into the complexity of the CD28 costimulatory pathway of T cells and will help to identify potential targets of therapeutic interventions for modulating anti-tumor T cell activation.

Antigens, CD↗

PubMed2005

A novel strategy for quantitative proteomics using isotope-coded protein labels.

Stable isotope labelling in combination with mass spectrometry has emerged as a powerful tool to identify and relatively quantify thousands of proteins within complex protein mixtures. Here we describe a novel method, termed isotope-coded protein label (ICPL), which is capable of high-throughput quantitative proteome profiling on a global scale. Since ICPL is based on stable isotope tagging at the frequent free amino groups of isolated intact proteins, it is applicable to any protein sample, including extracts from tissues or body fluids, and compatible to all separation methods currently employed in proteome studies. The method showed highly accurate and reproducible quantification of proteins and yielded high sequence coverage, indispensable for the detection of post-translational modifications and protein isoforms. The efficiency (e.g. accuracy, dynamic range, sensitivity, speed) of the approach is demonstrated by comparative analysis of two differentially spiked proteomes.

Caseins↗

PubMed2005

Identification of fatty acid binding proteins as markers associated with the initiation and/or progression of renal cell carcinoma.

Renal cell carcinoma (RCC) representing the most common neoplasia of the kidney in Western countries is a histologic diverse disease with an often unpredictable course. The prognosis of RCC is worsened with the onset of metastasis, and the therapies currently available are of limited success for the treatment of metastatic RCC. Although gene expression analyses and other methods are promising tools clarifying and standardizing the pathological classification of RCC, novel innovative molecular markers for the diagnosis, prognosis, and for the monitoring of this disease during therapy as well as potential therapeutic targets are urgently needed. Using proteome-based strategies, a number of RCC-associated markers either over-expressed or down-regulated in tumor lesions in comparison to the normal epithelium have been identified which have been implicated in tumorigenesis, but never linked to the initiation and/or progression of RCC. These include members of the fatty acid binding protein family, which have the potential to serve as diagnostic or prognostic markers for the screening of RCC patients.

Biomarkers, Tumor↗

PubMed2005

PorH, a new channel-forming protein present in the cell wall of Corynebacterium efficiens and Corynebacterium callunae.

Corynebacterium callunae and Corynebacterium efficiens are close relatives of the glutamate-producing mycolata species Corynebacterium glutamicum. The properties of the pore-forming proteins, extracted by organic solvents, were studied. The cell extracts contained channel-forming proteins that formed ion-permeable channels with a single-channel conductance of about 2 to 3 nS in 1 M KCl in a lipid bilayer assay. The corresponding proteins from both corynebacteria were purified to homogeneity and were named PorH(C.call) and PorH(C.eff). Electrophysiological studies of the channels suggested that they are wide and water-filled. Channels formed by PorH(C.call) are cation-selective, whereas PorH(C.eff) forms slightly anion-selective channels. Both proteins were partially sequenced. A multiple sequence alignment search within the known chromosome of C. efficiens demonstrated that it contains a gene that fits the partial amino acid sequence of PorH(C.eff). PorH(C.call) shows high homology to PorH(C.eff). PorH(C.eff) is encoded in the bacterial chromosome by a gene that is localized within the vicinity of the porA gene of C. efficiens. PorH(C.eff) has no signal sequence at the N terminus, which means that it is not exported by the Sec-secretion pathway. The structure of PorH in the cell wall of the corynebacteria is discussed.

Bacterial Outer Membrane Proteins↗

PubMed2005

The pyruvate kinase of Stigmatella aurantiaca is an indole binding protein and essential for development.

Myxospore formation of the myxobacterium Stigmatella aurantiaca can be uncoupled from the cooperative development i.e. fruiting body formation, by low concentrations of indole. Two putative indole receptor proteins were isolated by their capacity to bind indole and identified as pyruvate kinase (PK) and aldehyde dehydrogenase. The PK activity of Stigmatella crude extracts was stimulated by indole. Cloning of the PK gene (pykA) and the construction of a pykA disruption mutant strikingly revealed that PK is essential for multicellular development: Fruiting body formation was abolished in the mutant strain and indole-induced spore formation was delayed. The developmental defects could be complemented by insertion of the pykA gene at the mtaB locus of the Stigmatella genome excluding any polar effects of the pykA disruption.

Bacterial Proteins↗

PubMed2004

cAMP-dependent tyrosine phosphorylation of subunit I inhibits cytochrome c oxidase activity.

Signaling pathways targeting mitochondria are poorly understood. We here examine phosphorylation by the cAMP-dependent pathway of subunits of cytochrome c oxidase (COX), the terminal enzyme of the electron transport chain. Using anti-phospho antibodies, we show that cow liver COX subunit I is tyrosinephosphorylated in the presence of theophylline, a phosphodiesterase inhibitor that creates high cAMP levels, but not in its absence. The site of phosphorylation, identified by mass spectrometry, is tyrosine 304 of COX catalytic subunit I. Subunit I phosphorylation leads to a decrease of V(max) and an increase of K(m) for cytochrome c and shifts the reaction kinetics from hyperbolic to sigmoidal such that COX is fully or strongly inhibited up to 10 mum cytochrome c substrate concentrations, even in the presence of allosteric activator ADP. To assess our findings with the isolated enzyme in a physiological context, we tested the starvation signal glucagon on human HepG2 cells and cow liver tissue. Glucagon leads to COX inactivation, an effect also observed after incubation with adenylyl cyclase activator forskolin. Thus, the glucagon receptor/G-protein/cAMP pathway regulates COX activity. At therapeutic concentrations used for asthma relief, theophylline causes lung COX inhibition and decreases cellular ATP levels, suggesting a mechanism for its clinical action.

Allosteric Regulation↗

PubMed2004

Protein stoichiometry of a multiprotein complex, the human spliceosomal U1 small nuclear ribonucleoprotein: absolute quantification using isotope-coded tags and mass spectrometry.

The human U1 snRNP (small nuclear ribonucleoprotein), which is a part of the spliceosome, consists of U1 snRNA and ten different proteins: seven Sm proteins B/B', D1, D2, D3, E, F, and G and the three U1-specific proteins U1-70 K, U1-A, U1-C. To determine the stoichiometry of all ten proteins, the complex was denatured, digested completely with an endoproteinase and labeled with an amine-specific tag. Corresponding peptides were synthesized and labeled with the same tag containing heavier isotopes. The digest was then spiked with defined amounts of the synthetic peptides, and the resulting isotopic peptide pairs were analyzed quantitatively by mass spectrometry. The mass spectra provided information about the absolute amount of each component in the starting protein mixture. The use of the isotope-coded, amine-specific reagents propionyl-N-oxysuccinimide and nicotinoyl-N-oxysuccinimide was evaluated for stoichiometry determination; the nicotinoyl reagent was found to be advantageous because of its greater mass spectrometric sensitivity. Absolute quantities of all ten proteins were measured, showing equal numbers of all ten proteins in the U1 spliceosomal snRNP. These data demonstrate that quantitative mass spectrometry has great potential for the determination of the stoichiometry of multiprotein complexes.

Chromatography, Liquid↗

PubMed2004

Aminopeptidase N (CD13) is a molecular target of the cholesterol absorption inhibitor ezetimibe in the enterocyte brush border membrane.

Intestinal cholesterol absorption is an important regulator of serum cholesterol levels. Ezetimibe is a specific inhibitor of intestinal cholesterol absorption recently introduced into medical practice; its mechanism of action, however, is still unknown. Ezetimibe neither influences the release of cholesterol from mixed micelles in the gut lumen nor the transfer of cholesterol to the enterocyte brush border membrane. With membrane-impermeable Ezetimibe analogues we could demonstrate that binding of cholesterol absorption inhibitors to the brush border membrane of small intestinal enterocytes from the gut lumen is sufficient for inhibition of cholesterol absorption. A 145-kDa integral membrane protein was identified as the molecular target for cholesterol absorption inhibitors in the enterocyte brush border membrane by photoaffinity labeling with photoreactive Ezetimibe analogues (Kramer, W., Glombik, H., Petry, S., Heuer, H., Schafer, H. L., Wendler, W., Corsiero, D., Girbig, F., and Weyland, C. (2000) FEBS Lett. 487, 293-297). The 145-kDa Ezetimibe-binding protein was purified by three different methods and sequencing revealed its identity with the membrane-bound ectoenzyme aminopeptidase N ((alanyl)aminopeptidase; EC 3.4.11.2; APN; leukemia antigen CD13). The enzymatic activity of APN was not influenced by Ezetimibe (analogues). The uptake of cholesterol delivered by mixed micelles by confluent CaCo-2 cells was partially inhibited by Ezetimibe and nonabsorbable Ezetimibe analogues. Preincubation of confluent CaCo-2 cells with Ezetimibe led to a strong decrease of fluorescent APN staining with a monoclonal antibody in the plasma membrane. Independent on its enzymatic activity, aminopeptidase N is involved in endocytotic processes like the uptake of viruses. Our findings suggest that binding of Ezetimibe to APN from the lumen of the small intestine blocks endocytosis of cholesterol-rich membrane microdomains, thereby limiting intestinal cholesterol absorption.

Absorption↗

PubMed2004

Purification and partial amino acid sequences of the enzyme vinorine synthase involved in a crucial step of ajmaline biosynthesis.

The acetyl-CoA-dependent enzyme vinorine synthase was isolated from hybrid cell suspension cultures of Rauvolfia serpentina and Rhazya stricta. The sarpagan-type alkaloid gardneral was used as a substrate of the enzyme leading to the ajmalan-type 10-methoxyvinorine. An HPLC-based assay was developed to monitor vinorine synthase activity, which allowed establishing a five step purification procedure combining anion exchange, hydrophobic interaction, hydroxyapatite and gel filtration. Purification resulted in a yield of 0.2% and an approximately 991-fold enrichment of the acetyltransfer activity. SDS-PAGE analysis showed a Mr for the enzyme of approximately 50 kDa. The four peptide fragments generated by proteolysis of the pure enzyme with endoproteinase LysC and the N-terminal part of the enzyme were sequenced. The enzyme preparation (> 875-fold enrichment) delivering the N-terminal sequence was isolated from R. serpentina cell suspensions. Sequence alignment of the five peptides showed highest homologies in a range of 30-71% to acetyltransferases from other higher plants involved in natural plant product biosynthesis. Based on the partial sequences vinorine synthase is probably a novel member of the BAHD enzyme super family.

Acetyl-CoA C-Acetyltransferase↗