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Sabine Seitz

Publications and source records attributed to Sabine Seitz.

7 recordsLinked to original sources
PubMed2006

Reconstitution of paired T cell receptor alpha- and beta-chains from microdissected single cells of human inflammatory tissues.

We describe a strategy to "revive" putatively pathogenic T cells from frozen specimens of human inflammatory target organs. To distinguish pathogenic from irrelevant bystander T cells, we focused on cells that were (i) clonally expanded and (ii) in direct morphological contact with a target cell. Using CDR3 spectratyping, we identified clonally expanded T cell receptor (TCR) beta-chains in muscle sections of patients with inflammatory muscle diseases. By immunohistochemistry, we identified those Vbeta-positive T cells that fulfilled the morphological criteria of myocytotoxicity and isolated them by laser microdissection. Next, we identified coexpressed pairs of TCR alpha- and beta-chains by a multiplex PCR protocol, which allows the concomitant amplification of both chains from single cells. This concomitant amplification had not been achieved previously in histological sections, mainly because of the paucity of available anti-alpha-chain antibodies and the great heterogeneity of the alpha-chain genes. From muscle tissue of a patient with polymyositis, we isolated 64 T cells that expressed an expanded Vbeta1 chain. In 23 of these cells, we identified the corresponding alpha-chain. Twenty of these 23 alpha-chains were identical, suggesting antigen-driven selection. After functional reconstitution of the alphabeta-pairs, their antigen-recognition properties could be studied. Our results open avenues for combined analysis of the full TCR alpha- and beta-chain repertoire in human inflammatory tissues.

Autoimmunity↗
PubMed2006

Maltose and maltodextrin utilization by Bacillus subtilis.

Bacillus subtilis can utilize maltose and maltodextrins that are derived from polysaccharides, like starch or glycogen. In this work, we show that maltose is taken up by a member of the phosphoenolpyruvate-dependent phosphotransferase system and maltodextrins are taken up by a maltodextrin-specific ABC transporter. Uptake of maltose by the phosphoenolpyruvate-dependent phosphotransferase system is mediated by maltose-specific enzyme IICB (MalP; synonym, GlvC), with an apparent K(m) of 5 microM and a V(max) of 91 nmol . min(-1) . (10(10) CFU)(-1). The maltodextrin-specific ABC transporter is composed of the maltodextrin binding protein MdxE (formerly YvdG), with affinities in the low micromolar range for maltodextrins, and the membrane-spanning components MdxF and MdxG (formerly YvdH and YvdI, respectively), as well as the energizing ATPase MsmX. Maltotriose transport occurs with an apparent K(m) of 1.4 microM and a V(max) of 4.7 nmol . min(-1) . (10(10) CFU)(-1).

Bacillus subtilis↗
PubMed2006

YeeI, a novel protein involved in modulation of the activity of the glucose-phosphotransferase system in Escherichia coli K-12.

The membrane-bound protein EIICB(Glc) encoded by the ptsG gene is the major glucose transporter in Escherichia coli. This protein is part of the phosphoenolpyruvate:glucose-phosphotransferase system, a very important transport and signal transduction system in bacteria. The regulation of ptsG expression is very complex. Among others, two major regulators, the repressor Mlc and the cyclic AMP-cyclic AMP receptor protein activator complex, have been identified. Here we report identification of a novel protein, YeeI, that is involved in the regulation of ptsG by interacting with Mlc. Mutants with reduced activity of the glucose-phosphotransferase system were isolated by transposon mutagenesis. One class of mutations was located in the open reading frame yeeI at 44.1 min on the E. coli K-12 chromosome. The yeeI mutants exhibited increased generation times during growth on glucose, reduced transport of methyl-alpha-d-glucopyranoside, a substrate of EIICB(Glc), reduced induction of a ptsG-lacZ operon fusion, and reduced catabolite repression in lactose/glucose diauxic growth experiments. These observations were the result of decreased ptsG expression and a decrease in the amount of EIICB(Glc). In contrast, overexpression of yeeI resulted in higher expression of ptsG, of a ptsG-lacZ operon fusion, and of the autoregulated dgsA gene. The effect of a yeeI mutation could be suppressed by introducing a dgsA deletion, implying that the two proteins belong to the same signal transduction pathway and that Mlc is epistatic to YeeI. By measuring the surface plasmon resonance, we found that YeeI (proposed gene designation, mtfA) directly interacts with Mlc with high affinity.

Chromosomes, Bacterial↗
PubMed2005

The crystal structure of Mlc, a global regulator of sugar metabolism in Escherichia coli.

Mlc from Escherichia coli is a transcriptional repressor controlling the expression of a number of genes encoding enzymes of the phosphotransferase system (PTS), including ptsG and manXYZ, the specific enzyme II for glucose and mannose PTS transporters. In addition, Mlc controls the transcription of malT, the gene of the global activator of the mal regulon. The inactivation of Mlc as a repressor is mediated by binding to an actively transporting PtsG (EIICB(Glc)). Here we report the crystal structure of Mlc at 2.7 A resolution representing the first described structure of an ROK (repressors, open reading frames, and kinases) family protein. Mlc forms stable dimers thus explaining its binding affinity to palindromic operator sites. The N-terminal helix-turn-helix domain of Mlc is stabilized by the amphipathic C-terminal helix implicated earlier in EIICB(Glc) binding. Furthermore, the structure revealed a metal-binding site within the cysteine-rich ROK consensus motif that coordinates a structurally important zinc ion. A strongly reduced repressor activity was observed when two of the zinc-coordinating cysteine residues were exchanged against serine or alanine, demonstrating the role of zinc in Mlc-mediated repressor function. The structures of a putative fructokinase from Bacillus subtilis, the glucokinase from Escherichia coli, and a glucomannokinase from Arthrobacter sp. showed high structural homology to the ROK family part of Mlc.

Alanine↗
PubMed2004

The lamin B receptor of Drosophila melanogaster.

The lamin B receptor (LBR) is an integral membrane protein of the inner nuclear membrane that has so far been characterized only in vertebrates. Here, we describe the Drosophila melanogaster protein encoded by the annotated gene CG17952 that is the putative ortholog to the vertebrate LBR. The Drosophila lamin B receptor (dLBR) has the following properties in common with the vertebrate LBR. First, structure predictions indicate that the 741 amino acid dLBR protein possesses a highly charged N-terminal domain of 307 amino acids followed by eight transmembrane segments in the C-terminal domain of the molecule. Second, immunolocalization and cell fractionation reveal that the dLBR is an integral membrane protein of the inner nuclear membrane. Third, dLBR can be shown by co-immunoprecipitations and in vitro binding assays to bind to the Drosophila B-type lamin Dm0. Fourth, the N-terminal domain of dLBR is sufficient for in vitro binding to sperm chromatin and lamin Dm0. In contrast to the human LBR, dLBR does not possess sterol C14 reductase activity when it is expressed in the Saccharomyces cerevisiae erg24 mutant, which lacks sterol C14 reductase activity. Our data raise the possibility that, during evolution, the enzymatic activity of this insect protein had been lost. To determine whether the dLBR is an essential protein, we depleted it by RNA interference in Drosophila embryos and in cultured S2 and Kc167 cells. There is no obvious effect on the nuclear architecture or viability of treated cells and embryos, whereas the depletion of Drosophila lamin Dm0 in cultured cells and embryos caused morphological alterations of nuclei, nuclear fragility and the arrest of embryonic development. We conclude that dLBR is not a limiting component of the nuclear architecture in Drosophila cells during the first 2 days of development.

Amino Acid Sequence↗
PubMed2003

Analysis of the interaction between the global regulator Mlc and EIIBGlc of the glucose-specific phosphotransferase system in Escherichia coli.

Mlc is a global regulator acting as a transcriptional repressor for several genes and operons of Escherichia coli encoding sugar-metabolizing enzymes and uptake systems. The repressing activity of Mlc is inactivated by binding to the dephosphorylated form of EIICB(Glc) (PtsG), which is formed during the transport of glucose. Here, we demonstrate that EIIB(Glc), the cytoplasmic domain of PtsG, alone is sufficient to inactivate Mlc but only when EIIB(Glc) is attached to the membrane by a protein anchor, which can be unrelated to PtsG. Several EIIB(Glc) mutants, which were altered in and around the phosphorylation site (Cys-421) of EIIB(Glc), were tested for their ability to bind Mlc and to affect transcriptional repression by Mlc. The exchange of Cys-421 with serine or aspartate still allowed binding to Mlc, and in addition, derepression became constitutive, i.e. independent of phosphoenolpyruvate-dependent phosphotransferase system (PTS) phosphorylation. Mutations were made in the surface-exposed residues in the vicinity of Cys-421 and identified Arg-424 as essential for binding to Mlc. Binding of Mlc to the EIIB(Glc) constructs in membrane preparations paralleled their ability to derepress Mlc-dependent transcription in vivo. These observations demonstrate that it is not the charge change at Cys-421, produced by PTS phosphorylation, that allows Mlc binding but rather the structural change in the environment surrounding Cys-421 that the phosphorylation provokes. Native Mlc exists as a tetramer. Deleting 18 amino acids from the C-terminal removes a putative amphipathic helix and results in dimeric Mlc that is no longer able to repress.

Binding Sites↗