Biomedical subjects

R Kraft

Publications and source records attributed to R Kraft.

At least 19 recordsLinked to original sources

PubMed2001

Identification of nucleolin as a new L-selectin ligand.

Apart from leucocyte-endothelial interactions, the adhesion molecule L-selectin mediates the homotypic adhesion of leucocytes during recruitment at sites of acute inflammation, as well as intercellular adhesion of haematopoietic progenitor cells during haematopoiesis. There is evidence that, in addition to P-selectin glycoprotein ligand-1, other as-yet-unidentified proteins function as L-selectin ligands on human leucocytes and haematopoietic progenitor cells. In the present study, we show: (i) by affinity chromatography on L-selectin-agarose; (ii) by protein identification using MS; and (iii) by covalent cell-surface labelling with sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate that the multifunctional nuclear protein nucleolin is partly exposed on the cell surface, and is a ligand of L-selectin in human leucocytes and haematopoietic progenitor cells.

Amino Acid Sequence↗

PubMed2001

Targeting of the transcription factor Max during apoptosis: phosphorylation-regulated cleavage by caspase-5 at an unusual glutamic acid residue in position P1.

Max is the central component of the Myc/Max/Mad network of transcription factors that regulate growth, differentiation and apoptosis. Whereas the Myc and Mad genes and proteins are highly regulated, Max expression is constitutive and no post-translational regulation is known. We have found that Max is targeted during Fas-induced apoptosis. Max is first dephosphorylated and subsequently cleaved by caspases. Two specific cleavage sites for caspases in Max were identified, one at IEVE(10) decreasing S and one at SAFD(135) decreasing G near the C-terminus, which are cleaved in vitro by caspase-5 and caspase-7 respectively. Mutational analysis indicates that both sites are also used in vivo. Thus Max represents the first caspase-5 substrate. The unusual cleavage after a glutamic acid residue is observed only with full-length, DNA-binding competent Max protein but not with corresponding peptides, suggesting that structural determinants might be important for this activity. Furthermore, cleavage by caspase-5 is inhibited by the protein kinase CK2-mediated phosphorylation of Max at Ser-11, a previously mapped phosphorylation site in vivo. These findings suggest that Fas-mediated dephosphorylation of Max is required for cleavage by caspase-5. The modifications that occur on Max in response to Fas signalling affect the DNA-binding activity of Max/Max homodimers. Taken together, our findings uncover three distinct processes, namely dephosphorylation and cleavage by caspase-5 and caspase-7, that target Max during Fas-mediated apoptosis, suggesting the regulation of the Myc/Max/Mad network through its central component.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Importin 13: a novel mediator of nuclear import and export.

Importin beta-related receptors mediate translocation through nuclear pore complexes. Co-operation with the RanGTPase system allows them to bind and subsequently release their substrates on opposite sides of the nuclear envelope, which in turn ensures a directed nucleocytoplasmic transport. Here we identify a novel family member from higher eukaryotes that functions primarily, but not exclusively, in import. It accounts for nuclear accumulation of the SUMO-1/sentrin-conjugating enzyme hUBC9 and mediates import of the RBM8 (Y14) protein, and is therefore referred to as importin 13 (Imp13). Unexpectedly, Imp13 also shows export activity towards the translation initiation factor eIF1A and is thus a case where a single importin beta-like receptor transports different substrates in opposite directions. However, Imp13 operates differently from typical exportins in that the binding of eIF1A to Imp13 is only regulated indirectly by RanGTP, and the cytoplasmic release of eIF1A from Imp13 is triggered by the loading of import substrates onto Imp13.

Cell Nucleus↗

PubMed2001

COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system.

In higher eukaryotic cells, the p53 protein is degraded by the ubiquitin-26S proteasome system mediated by Mdm2 or the human papilloma virus E6 protein. Here we show that COP9 signalosome (CSN)-specific phosphorylation targets human p53 to ubiquitin-26S proteasome-dependent degradation. As visualized by electron microscopy, p53 binds with high affinity to the native CSN complex. p53 interacts via its N-terminus with CSN subunit 5/Jab1 as shown by far-western and pull-down assays. The CSN-specific phosphorylation sites were mapped to the core domain of p53 including Thr155. A phosphorylated peptide, Deltap53(145-164), specifically inhibits CSN-mediated phosphorylation and p53 degradation. Curcumin, a CSN kinase inhibitor, blocks E6-dependent p53 degradation in reticulocyte lysates. Mutation of Thr155 to valine is sufficient to stabilize p53 against E6-dependent degradation in reticulocyte lysates and to reduce binding to Mdm2. The p53T155V mutant accumulates in both HeLa and HL 60 cells and exhibits a mutant (PAb 240+) conformation. It induces the cyclin-dependent inhibitor p21. In HeLa and MCF-7 cells, inhibition of CSN kinase by curcumin or Deltap53(145-164) results in accumulation of endogenous p53.

Amino Acid Sequence↗

PubMed2001

Serum deprivation and NGF induce and modulate voltage-gated Na(+) currents in human astrocytoma cell lines.

Glial tumor cells derived from primary tissue express large voltage-gated Na(+) currents, whereas glioma cell lines usually lack this feature. We studied the effect of serum deprivation on the expression of Na(+) currents in two astrocytoma cell lines (1321N1 and A172). Serum deprivation for more than 2 days sufficed to induce large Na(+) currents in both cell lines; 300 nM of the specific blocker of voltage-gated Na(+) channels, tetrodotoxin, blocked these currents by about 85%. During serum deprivation, the cells also underwent morphological changes that were characterized by cell rounding and outgrowth of processes. Treatment with 100 ng/ml nerve growth factor (NGF) promoted these morphological changes and also accelerated the development of Na(+) currents. In 1321N1 cells, NGF increased the Na(+) current density after short serum deprivation (3--6 d) and changed several gating properties after longer serum deprivation (9--13 d). In comparison with cells from the early culture stage (3--6 d), the steady-state inactivation of the Na(+) current was shifted by -24 mV in NGF-treated cells from the late (9--13 d) culture stage. In untreated cells, this shift was only -13 mV. NGF accelerated the kinetics of inactivation and shifted the current-voltage relationship in cells from the late culture stage by -14 mV. In A172 cells, most of these effects were present already after short serum deprivation either in presence or absence of NGF. It is concluded that in astrocytoma cells, Na(+) currents are induced by serum deprivation and are modulated by NGF. This result supports the idea that NFG controls Na(+) currents in these cells by autocrine stimulation.

Astrocytoma↗

PubMed2001

The Rossmann fold of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a nuclear docking site for antisense oligonucleotides containing a TAAAT motif.

The subcellular localisation of oligodeoxynucleotides (ODN) is a major limitation for their use against nuclear targets. In this study we demonstrate that an antisense ODN directed against cytosolic phospholipase A(2) (cPLA2) mRNA is efficiently taken up and accumulates in the nuclei of endothelial cells (HUVEC), human monocytes and HeLa cells. Gel shift experiments and incubation of cells with oligonucleotide derivatives show that the anti-cPLA2 oligo binds a 37 kDa protein in nuclear extracts. The TAAAT sequence was identified as the major binding motif for the nuclear protein in competition experiments with mutated ODNs. Modification of the AAA triplet resulted in an ODN which failed to localise in the nucleus. Moreover, inserting a TAAAT motif into an ODN localising in the cytosol did not modify its localisation. The 37 kDa protein was purified and identified after peptide sequencing as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It was shown by confocal microscopy that GAPDH co-localises with anti-cPLA2 ODN in the nucleus and commercial GAPDH effectively binds the oligo. Competition experiments with increasing concentration of NAD(+) co-factor indicate that the GAPDH Rossmann fold is a docking site for antisense oligonucleotides containing a TAAAT motif.

Amino Acid Sequence↗

PubMed2000

Identification of two novel RanGTP-binding proteins belonging to the importin beta superfamily.

Nucleo-cytoplasmic transport comprises a large number of distinct pathways, many of which are defined by members of the importin beta superfamily of nuclear transport receptors. These transport receptors all directly interact with RanGTP to modulate the compartment-specific binding of their transport substrates. To identify new members of the importin beta family, we used affinity chromatography on immobilized RanGTP and isolated Ran-binding protein (RanBP) 16 from HeLa cell extracts. RanBP16 and its close human homologue, RanBP17, are distant members of the importin beta family. Like the other members of the transport receptor superfamily, RanBP16 interacts with the nuclear pore complex and is able to enter the nucleus independent of energy and additional nuclear transport receptors.

Amino Acid Sequence↗

PubMed2000

Interaction between interferon consensus sequence-binding protein and COP9/signalosome subunit CSN2 (Trip15). A possible link between interferon regulatory factor signaling and the COP9/signalosome.

Interferon consensus sequence-binding protein (ICSBP) is a member of the interferon regulatory factors (IRF) that has a pivotal role in mediating resistance to pathogenic infections in mice and in promoting the differentiation of myeloid cells. ICSBP exerts some of its transcriptional activities via association with other factors that enable its binding to a variety of promoters containing DNA composite elements. These interactions are mediated through a specific COOH-terminal domain termed IAD (IRF association domain). To gain a broader insight of the capacity of ICSBP to interact with other factors, yeast two-hybrid screens were performed using ICSBP-IAD as a bait against a B-cell cDNA library. Trip15 was identified as a specific interacting factor with ICSBP in yeast cells, which was also confirmed by in vitro glutathione S-transferase pull-down assays and by coimmunoprecipitation studies in COS7 cells. Trip15 was recently identified as a component of the COP9/signalosome (CSN) complex composed of eight evolutionary conserved subunits and thus termed CSN2. This complex has a role in cell-signaling processes, which is manifested by its associated novel kinase activity and by the involvement of its subunits in regulating multiple cell-signaling pathways and cell-cycle progression. We show that in vitro association of ICSBP with the CSN leads to phosphorylation of ICSBP at a unique serine residue within its IAD. The phosphorylated residue is essential for efficient association with IRF-1 and thus for the repressor activity of ICSBP exerted on IRF-1. This suggests that the CSN has a role in integrating incoming signals that affect the transcriptional activity of ICSBP.

3T3 Cells↗

PubMed2000

Deuterium isotope effects on (13)C NMR chemical shifts reflect the smaller steric size of CD(3) compared to CH(3) groups

A CD(3) group close in space to (but many bonds distant from) a carbon atom A causes a substituent effect on the chemical shift of C(A) that is algebraically smaller than the effect of a CH(3) group, in agreement with the notion of shorter C-D relative to C-H bonds. Hence, the deuterium isotope effect of CD(3) upon delta(C(A)) is shielding when the substituent effect is deshielding, and vice versa.

Journal Article↗

PubMed2000

Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes.

Transport receptors of the importin beta superfamily account for many of the nuclear import and export events in eukaryotic cells. They mediate translocation through nuclear pore complexes, shuttle between nucleus and cytoplasm and co-operate with the RanGTPase system to regulate their interactions with cargo molecules in a compartment-specific manner. We used affinity chromatography on immobilized RanGTP to isolate further candidate nuclear transport receptors and thereby identified exportin 4 as the most distant member of the importin beta family so far. Exportin 4 appears to be conserved amongst higher eukaryotes, but lacks obvious orthologues in yeast. It mediates nuclear export of eIF-5A (eukaryotic translation initiation factor 5A) and possibly that of other cargoes. The export signal in eIF-5A appears to be complex and to involve the hypusine modification that is unique to eIF-5A. We discuss possible cellular roles for nuclear export of eIF-5A.

Amino Acid Sequence↗

PubMed2000

Characterisation of the newly identified human Ump1 homologue POMP and analysis of LMP7(beta 5i) incorporation into 20 S proteasomes.

Biogenesis of mammalian 20 S proteasomes occurs via precursor complexes containing alpha and unprocessed beta subunits. A human homologue of the yeast proteasome maturation factor Ump1 was identified in 2D gels of 16 S precursor preparations and designated as POMP (proteasome maturation protein). We show that POMP is detected only in precursor fractions and not in fractions containing mature 20 S proteasome. Northern blot experiments revealed that expression of POMP is induced after treatment with interferon gamma. To analyse the role of the beta 5 propeptide for proper maturation and incorporation of the beta 5 subunit into the complex, human T2 cells, which highly express derivatives of the beta 5i subunit (LMP7), were studied. In contrast to yeast, the presence of the beta 5 propeptide is not essential for incorporation of LMP7 into the proteasome complex. Mutated LMP7 subunits either carrying the prosequence of beta 2i (LMP2) or containing a mutation in the active threonine site are incorporated like wild-type LMP7, while a LMP7 derivative lacking the prosequence completely is incorporated to a lesser extent. Although the absence of the prosequence does not affect incorporation of LMP7, its deletion leads to delayed proteasome maturation and thereby to an accumulation of precursor complexes. As a result of the precursor accumulation, an increased amount of the POMP protein can be detected in these cells.

Amino Acid Sequence↗

PubMed2000

Overexpression of the proteasome subunits LMP2, LMP7, and MECL-1, but not PA28 alpha/beta, enhances the presentation of an immunodominant lymphocytic choriomeningitis virus T cell epitope.

The proteasome is a large protease complex that generates most of the peptide ligands of MHC class I molecules either in their final form or in the form of N-terminally extended precursors. Upon the stimulation of cells with IFN-gamma, three constitutively expressed subunits of the 20S proteasome are replaced by the inducible subunits LMP2 (low-molecular mass polypeptide 2), LMP7, and MECL-1 (multicatalytic endopeptidase complex-like-1) to form so-called immunoproteasomes. We show in this study that overexpression of these three subunits in triple transfectants led to a marked enhancement in the H-2Ld-restricted presentation of the immunodominant nonameric epitope NP118, which is derived from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. Overexpression of the alpha and beta subunits of the IFN-gamma-inducible proteasome regulator PA28, in contrast, did not have a comparable effect. In vitro, immunoproteasomes as compared with constitutive proteasomes generated higher amounts of 11- and 12-mer fragments containing the NP118 epitope. These are likely to be cytosolic precursors of NP118, as a proline anchor residue in the second position of NP118 may interfere with TAP-mediated transport of the nonameric epitope itself. In conclusion, we provide evidence that up-regulation of the three inducible subunits, LMP2, LMP7, and MECL-1, can result in a marked improvement of Ag presentation and that, depending on the epitope, PA28 and immunoproteasomes may differentially affect Ag processing.

Adjuvants, Immunologic↗

PubMed2000

Voltage- and gamma-aminobutyric acid-activated membrane currents in the human medulloblastoma cell line MHH-MED-3.

The whole-cell patch clamp technique was used to characterize voltage- and neurotransmitter-activated currents in the medulloblastoma cell line MHH-MED-3 and cells from tissue slices and primary cultures of two medulloblastoma biopsies. These preparations revealed similar electrophysiological properties. All tested cells displayed 4-aminopyridine-sensitive delayed rectifying K(+) currents, gamma-aminobutyric acid(A) receptor-mediated Cl(-) currents and most of them inward rectifier K(+) currents. Transient inward currents were mainly carried by low-voltage activated T-type Ca(2+) channels in MHH-MED-3 cells, and tetrodotoxin-sensitive Na(+) channels in cells from the primary culture. From these characteristics we conclude that medulloblastoma cells share physiological features with developing cerebellar granule cells at an immature stage.

Adolescent↗

PubMed2000

Expression of the ALK protein by anaplastic large-cell lymphomas correlates with high proliferative activity.

A variable fraction of anaplastic large-cell lymphomas (ALCLs) exhibits a t(2;5)(p23;q35) translocation that results in expression of the chimeric hyperphosphorylated protein NPM-ALK (p80). Tumor cells expressing NPM-ALK exhibit markedly enhanced proliferative activity, but comparative cellular kinetic studies on ALK(+) (ALK lymphomas) and ALK(-) lymphomas are lacking. The present study showed that ALK(+) lymphomas, detected with the monoclonal antibody ALKc (n = 17), had significantly higher average values for the proliferation-associated parameters mitotic index, ana/telophase index, growth index (x x mitotic index - apoptotic index, assuming x = 3), percentages of Ki-67(+) cells and fraction of cells expressing cyclin A or B or the cell cycle-regulatory protein p34(cdc2) than did ALK(-) ALCLs (n = 15). Whether this intense proliferative activity contributes to the good response to chemotherapy and favorable outcome of ALK(+) ALCLs remains to be assessed in a larger series of patients. Our findings support the notion that ALK(+) and ALK(-) ALCLs are 2 distinct disease entities.

Adolescent↗

PubMed2000

The selective proteasome inhibitors lactacystin and epoxomicin can be used to either up- or down-regulate antigen presentation at nontoxic doses.

The complete inhibition of proteasome activities interferes with the production of most MHC class I peptide ligands as well as with cellular proliferation and survival. In this study we have investigated how partial and selective inhibition of the chymotrypsin-like activity of the proteasome by the proteasome inhibitors lactacystin or epoxomicin would affect Ag presentation. At 0.5-1 microM lactacystin, the presentation of the lymphocytic choriomeningitis virus-derived epitopes NP118 and GP33 and the mouse CMV epitope pp89-168 were reduced and were further diminished in a dose-dependent manner with increasing concentrations. Presentation of the lymphocytic choriomeningitis virus-derived epitope GP276, in contrast, was markedly enhanced at low, but abrogated at higher, concentrations of either lactacystin or epoxomicin. The inhibitor-mediated effects were thus epitope specific and did not correlate with the degradation rates of the involved viral proteins. Although neither apoptosis induction nor interference with cellular proliferation was observed at 0.5-1 microM lactacystin in vivo, this concentration was sufficient to alter the fragmentation of polypeptides by the 20S proteasome in vitro. Our results indicate that partial and selective inhibition of proteasome activity in vivo is a valid approach to modulate Ag presentation, with potential applications for the treatment of autoimmune diseases and the prevention of transplant rejection.

Acetylcysteine↗

PubMed2000

Mammalian Sec61 is associated with Sec62 and Sec63.

In yeast, efficient protein transport across the endoplasmic reticulum (ER) membrane may occur co-translationally or post-translationally. The latter process is mediated by a membrane protein complex that consists of the Sec61p complex and the Sec62p-Sec63p subcomplex. In contrast, in mammalian cells protein translocation is almost exclusively co-translational. This transport depends on the Sec61 complex, which is homologous to the yeast Sec61p complex and has been identified in mammals as a ribosome-bound pore-forming membrane protein complex. We report here the existence of ribosome-free mammalian Sec61 complexes that associate with two ubiquitous proteins of the ER membrane. According to primary sequence analysis both proteins display homology to the yeast proteins Sec62p and Sec63p and are therefore named Sec62 and Sec63, respectively. The probable function of the mammalian Sec61-Sec62-Sec63 complex is discussed with respect to its abundance in ER membranes, which, in contrast to yeast ER membranes, apparently lack efficient post-translational translocation activity.

Amino Acid Sequence↗

PubMed2000

Large conductance Ca(2+)-activated K(+) channels in human meningioma cells.

Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures. In whole cells, depolarization to voltages higher than +80 mV evoked a large K(+) outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC(50) = 5.3 mm). Raising the internal Ca(2+) from 10 nm to 2 mm shifted the voltage of half-maximum activation (V(1/2)) of the K(+) current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca(2+)-activated (BK(Ca)) K(+) channel with a conductance of 296 pS (130 mm K(+) at both sides of the patch). V(1/2) of single-channel currents was +6, -12, -46, and -68 mV in the presence of 1, 10, 100, and 1000 microm Ca(2+), respectively, at the internal face of the patch. In cell-attached patches the open probability (P(o)) of BK(Ca) channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K(+) currents with 10 nm Ca(2+) in the pipette. Application of 20 microm cytochalasin D increased P(o) of BK(Ca) channels in cell-attached patches within minutes. These data suggest that the activation of BK(Ca) channels in meningioma cells does not only depend on voltage and internal Ca(2+) but is also controlled by the cytoskeleton.

Aged↗

PubMed2000

Phosphorylation of Oct-2 at sites located in the POU domain induces differential down-regulation of Oct-2 DNA-binding ability.

We compared the effects of phosphorylation of Oct-2 protein on its binding to the consensus octamer sequence (ATGCAAAT) and two non-canonical sequences present in human (AAGCAAAT) and murine (AAACAAAT) promoters of the BLR1 (Burkitts' lymphoma receptor 1) gene encoding chemokine receptor CXCR5 (CXC-chemokine receptor 5). The latter cis-acting elements represent low-affinity recognition sequences for the octamer transcription factors. Okadaic acid was found to induce hyperphosphorylation of Oct-2 specifically in cells of lymphoid lineage. Potentially phosphorylated amino acid residues localized to the POU-specific domain of Oct-2. Whereas binding of Oct-2 to the octamer site from the human BLR1 promoter or to the consensus octamer sequence was unaffected by phosphorylation of this factor, a strong reduction of Oct-2 binding to the octamer site from the murine BLR1 promoter was observed. This finding correlates well with the down-regulation of expression of the BLR1 gene in murine splenic cells but not in lymphoid cells of human origin treated with okadaic acid. These data support the hypothesis that phosphorylation of Oct-2 may be a mechanism by which activities of the promoters containing non-canonical octamer sequences are differentially regulated in response to extracellular stimuli.

Amino Acid Sequence↗