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Biomedical subjects

P Henklein

Publications and source records attributed to P Henklein.

At least 19 recordsLinked to original sources
PubMed2004

PB1-F2, an influenza A virus-encoded proapoptotic mitochondrial protein, creates variably sized pores in planar lipid membranes.

A frameshifted region of the influenza A virus PB1 gene encodes a novel protein, termed PB1-F2, a mitochondrial protein that can induce cell death. Many proapoptotic proteins are believed to act at the mitochondrial outer membrane to form an apoptotic pore with lipids. We studied the interaction of isolated, synthetic PB1-F2 (sPB1-F2) peptide with planar phospholipid bilayer membranes. The presence of nanomolar concentrations of peptide in the bathing solution induced a transmembrane conductance that increased in a potential-dependent manner. Positive potential on the side of protein addition resulted in a severalfold increase in the rate of change of membrane conductance. sPB1-F2-treated membranes became permeable to monovalent cations, chloride, and to a lesser extent, divalent ions. Despite various experimental conditions, we did not detect the distinctive conductance levels typical of large, stable pores, protein channels, or even pores that are partially proteinaceous. Rather, membrane conductance induced by sPB1-F2 fluctuated and visited almost all conductance values. sPB1-F2 also dramatically decreased bilayer stability in an electric field, consistent with a decrease in the line tension of a lipidic pore. Since similar membrane-destabilizing profiles are seen with proapoptotic proteins (e.g., Bax) and the cytoplasmic helix of human immunodeficiency virus gp41, we suggest that the basis for sPB1-F2-induced cell death may be the permeabilization and destabilization of mitochondrial membranes, leading to macromolecular leakage and apoptosis.

Apoptosis↗
PubMed2002

PAR-1- and PAR-3-type thrombin receptor expression in primary cultures of human renal cell carcinoma cells.

In this study, we report coexpression of proteinase-activated receptor (PAR)-1- and PAR-3-type thrombin receptors in primary cultures obtained from surgically resected specimens of renal cell carcinomas (RCCs). Receptor expression on RNA level was evaluated by using the RT-PCR technique. Results demonstrated the presence of mRNA encoding PAR-1 and PAR-3, but mRNA encoding PAR-4 could not be found in human RCC cells. The expression of PAR-1 and PAR-3 on protein level was investigated with confocal laser fluorescence and freeze-fracture electron microscopy. Both thrombin receptor types were localized on the cell membrane but were also found on intracellular compartments of RCC cells. On the outer cell membrane, clustering of PAR-1 and PAR-3 molecules was partly observed. This is the first study demonstrating presence of both PAR-1 and PAR-3 in human carcinoma cells.

Calcium↗
PubMed2001

Identification of HLA-B27-restricted peptides from the Chlamydia trachomatis proteome with possible relevance to HLA-B27-associated diseases.

The association of HLA-B27 with ankylosing spondylitis and reactive arthritis is the strongest one known between an MHC class I Ag and a disease. We have searched the proteome of the bacterium Chlamydia trachomatis for HLA-B27 binding peptides that are stimulatory for CD8(+) cells both in a model of HLA-B27 transgenic mice and in patients. This was done by combining two biomathematical computer programs, the first of which predicts HLA-B27 peptide binding epitopes, and the second the probability of HLA-B27 peptide generation by the proteasome system. After preselection, immunodominant peptides were identified by Ag-specific flow cytometry. Using this approach we have identified for the first time nine peptides derived from different C. trachomatis proteins that are stimulatory for CD8(+) T cells. Eight of these nine murine-derived peptides were recognized by cytotoxic T cells. The same strategy was used to identify B27-restricted chlamydial peptides in three patients with reactive arthritis. Eleven peptides were found to be stimulatory for patient-derived CD8(+) T cells, of which eight overlapped those found in mice. Additionally, we applied the tetramer technology, showing that a B27/chlamydial peptide containing one of the chlamydial peptides stained CD8(+) T cells in patients with Chlamydia-induced arthritis. This comprehensive approach offers the possibility of clarifying the pathogenesis of B27-associated diseases.

Animals↗
PubMed2001

The solution structure and heme binding of the presequence of murine 5-aminolevulinate synthase.

The mitochondrial import of 5-aminolevulinate synthase (ALAS), the first enzyme of the mammalian heme biosynthetic pathway, requires the N-terminal presequence. The 49 amino acid presequence transit peptide (psALAS) for murine erythroid ALAS was chemically synthesized, and circular dichroism and (1)H nuclear magnetic resonance (NMR) spectroscopies used to determine structural elements in trifluoroethanol/H(2)O solutions and micellar environments. A well defined amphipathic alpha-helix, spanning L22 to F33, was present in psALAS in 50% trifluoroethanol. Further, a short alpha-helix, defined by A5-L8, was also apparent in the 26 amino acid N-terminus peptide, when its structure was determined in sodium dodecyl sulfate. Heme inhibition of ALAS mitochondrial import has been reported to be mediated through cysteine residues in presequence heme regulatory motifs (HRMs). A UV/visible and (1)H NMR study of hemin and psALAS indicated that a heme-peptide interaction occurs and demonstrates, for the first time, that heme interacts with the HRMs of psALAS.

5-Aminolevulinate Synthetase↗
PubMed2001

The murine cytomegalovirus pp89 immunodominant H-2Ld epitope is generated and translocated into the endoplasmic reticulum as an 11-mer precursor peptide.

The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitopes is known to be generated as precursor peptides requiring trimming either before or after translocation into the endoplasmic reticulum (ER). In this study, we have followed the proteasomal processing and TAP-dependent ER translocation of the immunodominant epitope of the murine CMV immediate early protein pp89. For the first time, we experimentally linked peptide generation by the proteasome system and TAP-dependent ER translocation. Our experiments show that the proteasome generates both an N-terminally extended 11-mer precursor peptide as well as the correct H2-L(d) 9-mer epitope, a process that is accelerated in the presence of PA28. Our direct peptide translocation assays, however, demonstrate that only the 11-mer precursor peptide is transported into the ER by TAPs, whereas the epitope itself is not translocated. In consequence, our combined proteasome/TAP assays show that the 11-mer precursor is the immunorelevant peptide product that requires N-terminal trimming in the ER for MHC class I binding.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

A novel PAR-1-type thrombin receptor signaling pathway: cyclic AMP-independent activation of PKA in SNB-19 glioblastoma cells.

Cellular effects of thrombin are mediated by members of a new subfamily of G protein-coupled receptors designated proteinase-activated receptors (PARs) with the prototype PAR-1. Investigation of PAR-1-induced signaling has been shown to be very important in clarifying thrombin's role in cell metabolism, differentiation, and growth. We evaluated connection of PAR-1 with the cAMP/PKA pathway in SNB-19 glioblastoma cells. Alpha-thrombin and the synthetic PAR-1 agonist SFLLRN stimulated PKA as shown by increased PKA activity and translocation of the catalytic PKA alpha subunits (PKA(cat)alpha) into the nucleus. However, no effect on cAMP could be observed. PKA(cat)alpha was found to be associated with nuclear factor-kappa B (NF-kappaB) p65 and its inhibitor protein IkappaB in SNB-19 cells. After PAR-1 stimulation, this association was markedly diminished. We conclude that PAR-1 mediates PKA activation without altering cAMP levels but includes NF-kappaB-associated PKA(cat)alpha in SNB-19 glioblastoma cells. This is the first evidence for a cAMP-independent PKA signaling by a G protein-coupled receptor.

Active Transport, 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

A novel influenza A virus mitochondrial protein that induces cell death.

While searching for alternative reading-frame peptides encoded by influenza A virus that are recognized by CD8+ T cells, we found an abundant immunogenic peptide encoded by the +1 reading frame of PB1. This peptide derives from a novel conserved 87-residue protein, PB1-F2, which has several unusual features compared with other influenza gene products in addition to its mode of translation. These include its absence from some animal (particularly swine) influenza virus isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation and mitochondrial localization. Exposure of cells to a synthetic version of PB1-F2 induces apoptosis, and influenza viruses with targeted mutations that interfere with PB1-F2 expression induce less extensive apoptosis in human monocytic cells than those with intact PB1-F2. We propose that PB1-F2 functions to kill host immune cells responding to influenza virus infection.

Amino Acid Sequence↗
PubMed2000

Requirement of specific intrahelical interactions for stabilizing the inactive conformation of glycoprotein hormone receptors.

Systematic analysis of structural changes induced by activating mutations has been frequently utilized to study activation mechanisms of G-protein-coupled receptors (GPCRs). In the thyrotropin receptor and the lutropin receptor (LHR), a large number of naturally occurring mutations leading to constitutive receptor activation were identified. Saturating mutagenesis studies of a highly conserved Asp in the junction of the third intracellular loop and transmembrane domain 6 suggested a participation of this anionic residue in a salt bridge stabilizing the inactive receptor conformation. However, substitution of all conserved cationic residues at the cytoplasmic receptor surface did not support this hypothesis. Asp/Glu residues are a common motif at the N-terminal ends of alpha-helices terminating and stabilizing the helical structure (helix capping). Since Asp/Glu residues in the third intracellular loop/transmembrane domain 6 junction are not only preserved in glycoprotein hormone receptors but also in other GPCRs we speculated that this residue probably participates in an N-terminal helix-capping structure. Poly-Ala stretches are known to form and stabilize alpha-helices. Herein, we show that the function of the highly conserved Asp can be mimicked by poly-Ala substitutions in the LHR and thyrotropin receptor. CD and NMR studies of peptides derived from the juxtamembrane portion of the LHR confirmed the helix extension by the poly-Ala substitution and provided further evidence for an involvement of Asp in a helix-capping structure. Our data implicate that in addition to well established interhelical interactions the inactive conformation of GPCRs is also stabilized by specific intrahelical structures.

Amino Acid Sequence↗
PubMed2000

Functional and structural characterization of synthetic HIV-1 Vpr that transduces cells, localizes to the nucleus, and induces G2 cell cycle arrest.

Human immunodeficiency virus (HIV) Vpr contributes to nuclear import of the viral pre-integration complex and induces G(2) cell cycle arrest. We describe the production of synthetic Vpr that permitted the first studies on the structure and folding of the full-length protein. Vpr is unstructured at neutral pH, whereas under acidic conditions or upon addition of trifluorethanol it adopts alpha-helical structures. Vpr forms dimers in aqueous trifluorethanol, whereas oligomers exist in pure water. (1)H NMR spectroscopy allows the signal assignment of N- and C-terminal amino acid residues; however, the central section of the molecule is obscured by self-association. These findings suggest that the in vivo folding of Vpr may require structure-stabilizing interacting factors such as previously described interacting cellular and viral proteins or nucleic acids. In biological studies we found that Vpr is efficiently taken up from the extracellular medium by cells in a process that occurs independent of other HIV-1 proteins and appears to be independent of cellular receptors. Following cellular uptake, Vpr is efficiently imported into the nucleus of transduced cells. Extracellular addition of Vpr induces G(2) cell cycle arrest in dividing cells. Together, these findings raise the possibility that circulating forms of Vpr observed in HIV-infected patients may exert biological effects on a broad range of host target cells.

Amino Acid Sequence↗
PubMed2000

Membrane interactions and alignment of structures within the HIV-1 Vpu cytoplasmic domain: effect of phosphorylation of serines 52 and 56.

The cytoplasmic domain of the HIV-1 accessory protein Vpu is involved in the binding and degradation of the viral receptor CD4. In order to analyze previous structural models in the context of membrane environments, regions of Vpu(CYTO) incorporating particular conformational features have been synthesized and labelled with (15)N at selected backbone amides. Well-oriented proton-decoupled (15)N solid-state NMR spectra with (15)N chemical shifts at the most upfield position indicate that the amphipathic helix within [(15)N-Leu 45]-Vpu(27-57) strongly interacts with mechanically aligned POPC bilayers and adopts an orientation parallel to the membrane surface. No major changes in the topology of this membrane-associated amphipathic helix were observed upon phosphorylation of serine residues 52 and 56, although this modification regulates biological function of Vpu. In contrast, [(15)N-Ala 62]-Vpu(51-81) exhibits a pronounced (15)N chemical shift anisotropy.

Human Immunodeficiency Virus Proteins↗
PubMed2000

Comparison of the effects of 5- and 6-HOAt on model peptide coupling reactions relative to the cases for the 4- and 7-Isomers.

Synthesis of 5- and 6-HOAt has completed the full set of the four HOAt isomers derived from HOBt by insertion of a single nitrogen atom in the benzenoid nucleus. Comparison of the reactivity of all four isomers in model peptide coupling reactions has confirmed the unique character of the 7-isomer in promoting selectivity and maintaining configuration at the reactive carboxylic acid residue.

Aniline Compounds↗
PubMed2000

Meizothrombin, an intermediate of prothrombin activation, stimulates human glioblastoma cells by interaction with PAR-1-type thrombin receptors.

Thrombin induces well-characterized effects on normal and neoplastic brain cells by interaction with protease-activated receptor (PAR)-type thrombin receptors. However, nothing is known about the function of intermediate enzymes of prothrombin activation recently shown to evoke PAR-1-mediated signaling in smooth muscle cells. Therefore, we investigated the effect of recombinant human meizothrombin (rMT), one of thrombin's catalytically active precursor enzymes in the prothrombin cleavage cascade, on calcium mobilization in human SNB-19 glioblastoma cells. By using reverse-transcription polymerase chain reaction, immunofluorescence studies with a monoclonal anti-PAR-1 antibody and calcium measurements, SNB-19 cells were shown to express functional PAR-1-type thrombin receptors. PAR-1 is not only a receptor for thrombin in SNB-19 cells but was also activated by rMT very effectively. Under the conditions used in our experiments, SNB-19 cells stimulated with thrombin after rMT challenge were unable to elicit a new calcium response and vice versa. In addition, both rMT and thrombin induced no further calcium signal after that observed with the PAR-1-activating peptide SFLLRN. Therefore, rMT and thrombin seem to activate calcium signaling by similar mechanisms including PAR-1. Our results demonstrate rMT as a potent activator of PAR-1-type thrombin receptors in SNB-19 glioblastoma cells, suggesting a function of catalytically active thrombin precursor enzymes in cells of glial origin.

Calcium↗
PubMed2000

Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin alpha and gamma subunits.

The role of the putative fourth cytoplasmic loop of rhodopsin in the binding and catalytic activation of the heterotrimeric G protein, transducin (G(t)), is not well defined. We developed a novel assay to measure the ability of G(t), or G(t)-derived peptides, to inhibit the photoregeneration of rhodopsin from its active metarhodopsin II state. We show that a peptide corresponding to residues 340-350 of the alpha subunit of G(t), or a cysteinyl-thioetherfarnesyl peptide corresponding to residues 50-71 of the gamma subunit of G(t), are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group from the gamma-derived peptide, prevents inhibition. Mutation of the amino-terminal region of the fourth cytoplasmic loop of rhodopsin affects interaction with G(t) (Marin, E. P., Krishna, A. G., Zvyaga T. A., Isele, J., Siebert, F., and Sakmar, T. P. (2000) J. Biol. Chem. 275, 1930-1936). Here, we provide evidence that this segment of rhodopsin interacts with the carboxyl-terminal peptide of the alpha subunit of G(t). We propose that the amino-terminal region of the fourth cytoplasmic loop of rhodopsin is part of the binding site for the carboxyl terminus of the alpha subunit of G(t) and plays a role in the regulation of betagamma subunit binding.

Amino Acid Sequence↗
PubMed2000

The two-receptor system PAR-1/PAR-4 mediates alpha-thrombin-induced [Ca(2+)](i) mobilization in human astrocytoma cells.

The proteinase-activated receptor 1 (PAR-1) was characterized as a functional receptor for thrombin in cells from different brain tumor entities. Whether PAR-1 alone accounts for thrombin-induced effects in human cancer cells, or whether other PAR contribute is unknown. We established primary cultures from two neurosurgically removed human astrocytomas and investigated intracellular signaling roles of PAR-1 and PAR-4 by estimating the effect of alpha-thrombin and PAR-activating peptides on [Ca(2+)](i) mobilization in single astrocytoma cells. alpha-Thrombin or the PAR-1-activating peptide SFLLRN induced a transient calcium mobilization. This suggests the involvement of PAR-1 in alpha-thrombin-induced calcium signaling in human astrocytoma cells. In addition, a second, PAR-4-dependent, mechanism exists. This was deduced from the findings that a further calcium signal could be observed in human astrocytoma cells stimulated with alpha-thrombin after SFLLRN and the PAR-4-activating peptide GYPGQV also induced a calcium response. In addition, the observation that trypsin, known to activate both PAR-2 and PAR-4, but not the specifically PAR-2-activating peptide SLIGRL induced calcium signaling is a further indication of functional PAR-4-type thrombin receptors in human astrocytoma cells. This is the first report demonstrating a signaling role for a dual thrombin receptor system in human tumor cells.

Apoptosis Regulatory Proteins↗
PubMed2000

In vitro pharmacological activity of the tetrahydroisoquinoline salsolinol present in products from Theobroma cacao L. like cocoa and chocolate.

Cocoa and chocolate contain the tetrahydroisoquinoline alkaloid salsolinol up to a concentration of 25 microg/g. Salsolinol is a dopaminergic active compound which binds to the D(2) receptor family, especially to the D(3) receptor with a K(i) of 0.48+/-0.021 micromol/l. It inhibits the formation of cyclic AMP and the release of beta-endorphin and ACTH in a pituitary cell system. Taking the detected concentration and the pharmacological properties into account, salsolinol seems to be one of the main psychoactive compounds present in cocoa and chocolate and might be included in chocolate addiction.

Adrenocorticotropic Hormone↗
PubMed2000

Different signaling pathways are involved in CCK(B) receptor-mediated MAP kinase activation in COS-7 cells.

Recently, the involvement of the MAP kinase ERK in mitogenic signaling of cholecystokininB (CCK(B)) receptors has been shown. However, the intracellular effector systems involved in this signaling pathway are poorly defined. In this study, we used COS-7 cells transiently transfected with the human CCK(B) receptor to investigate cholecystokinin-induced MAP kinase activation. CCK-8 induced activation of ERK2 which is associated with its phosphorylation and localization in the nucleus. The CCK-8-dependent ERK stimulation is sensitive to wortmannin an inhibitor of phosphoinositide 3-kinases (PI3Ks) indicating the involvement of PI3K activity. To identify the PI3K species involved in mitogenic signaling of the CCK(B) receptor several dominant-negative mutants of PI3K regulatory and catalytic subunits were transiently expressed. Surprisingly, different catalytically inactive mutants of the G protein-sensitive PI3Kgamma did not affect ERK stimulation induced by CCK, whereas a dominant-negative mutant of the regulatory p85 subunit induced significant inhibition of CCK-dependent ERK activity. These results indicate an involvement of PI3K class 1A species alpha, beta or/and delta in signal transduction via CCK(B) receptors. In addition, protein kinase C (PKC)-dependent signaling pathways contribute to CCK(B)-mediated MAP kinase signaling as shown by inhibition of CCK-8-induced ERK activation by the PKC inhibitor bisindolylmaleimide.

Animals↗