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Peter Henklein

Publications and source records attributed to Peter Henklein.

At least 19 recordsLinked to original sources
PubMed2006

Structure, dynamics and topology of membrane polypeptides by oriented 2H solid-state NMR spectroscopy.

Knowledge of the structure, dynamics and interactions of polypeptides when associated with phospholipid bilayers is key to understanding the functional mechanisms of channels, antibiotics, signal- or translocation peptides. Solid-state NMR spectroscopy on samples uniaxially aligned relative to the magnetic field direction offers means to determine the alignment of polypeptide bonds and domains relative to the bilayer normal. Using this approach the (15)N chemical shift of amide bonds provides a direct indicator of the approximate helical tilt, whereas the (2)H solid-state NMR spectra acquired from peptides labelled with 3,3,3-(2)H(3)-alanines contain valuable complimentary information for a more accurate analysis of tilt and rotation pitch angles. The deuterium NMR line shapes are highly sensitive to small variations in the alignment of the C(alpha)-C(beta) bond relative to the magnetic field direction and, therefore, also the orientational distribution of helices relative to the membrane normal. When the oriented membrane samples are investigated with their normal perpendicular to the magnetic field direction, the rate of rotational diffusion can be determined in a semi-quantitative manner and thereby the aggregation state of the peptides can be analysed. Here the deuterium NMR approach is first introduced showing results from model amphipathic helices. Thereafter investigations of the viral channel peptides Vpu(1-27) and Influenza A M2(22-46) are shown. Whereas the (15)N chemical shift data confirm the transmembrane helix alignments of these hydrophobic sequences, the deuterium spectra indicate considerable mosaic spread in the helix orientations. At least two peptide populations with differing rotational correlation times are apparent in the deuterium spectra of the viral channels suggesting an equilibrium between monomeric peptides and oligomeric channel configurations under conditions where solid-state NMR structural studies of these peptides have previously been performed.

Deuterium↗
PubMed2006

Structural characterization and oligomerization of PB1-F2, a proapoptotic influenza A virus protein.

Recently, a novel 87-amino acid influenza A virus protein with proapoptotic properties, PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most known human influenza A virus isolates. Here we characterize the molecular structure of a biologically active synthetic version of the protein (sPB1-F2). Western blot analysis, chemical cross-linking, and NMR spectroscopy afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization mediated by two distinct domains located in the N and C termini, respectively. CD and (1)H NMR spectroscopic analyses indicate that the stability of structured regions in the molecule clearly depends upon the hydrophobicity of the solvent. In aqueous solutions, the behavior of sPB1-F2 is typical of a largely random coil peptide that, however, adopts alpha-helical structure upon the addition of membrane mimetics. (1)H NMR analysis of three overlapping peptides afforded, for the first time, direct experimental evidence of the presence of a C-terminal region with strong alpha-helical propensity comprising amino acid residues Ile(55)-Lys(85) connected via an essentially random coil structure to a much weaker helix-like region, located in the N terminus between residues Trp(9) and Lys(20). The C-terminal helix is not a true amphipathic helix and is more compact than previously predicted. It corresponds to a positively charged region previously shown to include the mitochondrial targeting sequence of PB1-F2. The consequences of the strong oligomerization and helical propensities of the molecule are discussed and used to formulate a hypothetical model of its interaction with the mitochondrial membrane.

Amino Acid Sequence↗
PubMed2006

Rhodopsin-transducin coupling: role of the Galpha C-terminus in nucleotide exchange catalysis.

In the early steps of visual signal transduction, light-activated rhodopsin (R*) catalyzes GDP/GTP exchange in the heterotrimeric G protein (Galphabetagamma) transducin. We recently reported that the catalytic interaction involves two sequential steps. An initial docking between R* and Gbetagamma leads to conformational changes which make the C-terminus of Galpha (CTalpha) available for binding to R*. Binding of CTalpha by R* then triggers GDP/GTP exchange in the Galpha subunit. To further study this two-step mechanism, we investigated different single amino acid substitutions within CTalpha and discuss the effects of high affinity mutations on nucleotide exchange catalysis.

Amino Acid Substitution↗
PubMed2006

Effects of transgenic expression of HIV-1 Vpr on lipid and energy metabolism in mice.

HIV infection is associated with abnormal lipid metabolism, body fat redistribution, and altered energy expenditure. The pathogenesis of these complex abnormalities is unclear. Viral protein R (Vpr), an HIV-1 accessory protein, can regulate gene transcription mediated by the glucocorticoid receptor and peroxisome proliferator-activated receptor-gamma and affect mitochondrial function in vitro. To test the hypothesis that expression of Vpr in liver and adipocytes can alter lipid metabolism in vivo, we engineered mice to express Vpr under control of the phosphoenolpyruvate carboxykinase promoter in a tissue-specific and inducible manner and investigated the effects of dietary fat, indinavir, and dexamethasone on energy metabolism and body composition. The transgenic mice expressed Vpr mRNA in white and brown adipose tissues and liver and immunoaffinity capillary electrophoresis revealed that they had free Vpr protein in the plasma. Compared with wild-type (WT) animals, Vpr mice had lower plasma triglyceride levels after 6 wk (P < 0.05) but not after 10 wk of a high-fat diet and lower plasma cholesterol levels after 10 wk of high-fat diet (P < 0.05). Treatment with dexamethasone obviated group differences, whereas indinavir had no significant independent effect on lipids. In the fasted state, Vpr mice had a higher respiratory quotient than WT mice (P < 0.05). These data provide the first in vivo evidence that HIV-1 Vpr expressed at low levels in adipose tissues and liver can 1) circulate in the blood, 2) regulate lipid and fatty acid metabolism, and 3) alter fuel selection for oxidation in the fasted state.

Adipose Tissue↗
PubMed2006

Signal transfer from GPCRs to G proteins: role of the G alpha N-terminal region in rhodopsin-transducin coupling.

Catalysis of nucleotide exchange in heterotrimeric G proteins (Galphabetagamma) is a key step in cellular signal transduction mediated by G protein-coupled receptors. The Galpha N terminus with its helical stretch is thought to be crucial for G protein/activated receptor (R(*)) interaction. The N-terminal fatty acylation of Galpha is important for membrane targeting of G proteins. By applying biophysical techniques to the rhodopsin/transducin model system, we studied the effect of N-terminal truncations in Galpha. In Galphabetagamma, lack of the fatty acid and Galpha truncations up to 33 amino acids had little effect on R(*) binding and R(*)-catalyzed nucleotide exchange, implying that this region is not mandatory for R(*)/Galphabetagamma interaction. However, when the other hydrophobic modification of Galphabetagamma, the Ggamma C-terminal farnesyl moiety, is lacking, R(*) interaction requires the fatty acylated Galpha N terminus. This suggests that the two hydrophobic extensions can replace each other in the interaction of Galphabetagamma with R(*). We propose that in native Galphabetagamma, these two terminal regions are functionally redundant and form a microdomain that serves both to anchor the G protein to the membrane and to establish an initial docking complex with R(*). Accordingly, we find that the native fatty acylated Galpha is competent to interact with R(*) even in the absence of Gbetagamma, whereas nonacylated Galpha requires Gbetagamma for interaction. Experiments with N-terminally truncated Galpha subunits suggest that in the second step of the catalytic process, the receptor binds to the alphaN/beta1-loop region of Galpha to reduce nucleotide affinity and to make the Galpha C terminus available for subsequent interaction with R(*).

Amino Acid Sequence↗
PubMed2006

Identification of HIV-1 Tat peptides for future therapeutic angiogenesis.

Therapeutic angiogenesis represents a novel approach to treat critical limb ischemia when revascularization is no more an option. The clinical use of the vascular endothelial growth factor is questioned, because of its side effects. This study was designed to identify and characterize human immunodeficiency virus type 1 (HIV-1) Tat-derived peptides based on their pro-angiogenic properties. A series of Tat-derived peptides were synthesized containing mutations in the basic domain. To minimize side effects Tat peptides were selected exerting no effects on the proteasome and on the viability of human umbilical vein endothelial cells (HUVEC). Tatpep5, 15, and 16 increased the endogenous levels of the pro-angiogenic transcription factors c-Jun and SP-1 as well as the production of the plasminogen activator inhibitor-1 (PAI-1) by HUVEC. A significant induction of endothelial cell invasion was observed upon treatment of HUVEC with Tat peptides. In addition, selected Tat peptides induced tube formation by HUVEC as visualized and quantified in a Matrigel matrix. Our data demonstrate that the selected Tat peptides fulfill essential criteria for pro-angiogenic substances. They represent the basis for the development of novel pro-angiogenic drugs for future therapeutic angiogenesis, which might be applied for treatment of unreconstructible critical limb ischemia.

Angiogenesis Inducing Agents↗
PubMed2006

Sequence requirements and an optimization strategy for short antimicrobial peptides.

Short antimicrobial host-defense peptides represent a possible alternative as lead structures to fight antibiotic resistant bacterial infections. Bac2A is a 12-mer linear variant of the naturally occurring bovine host defense peptide, bactenecin, and demonstrates moderate, broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria as well as against the yeast Candida albicans. With the assistance of a method involving peptide synthesis on a cellulose support, the primary sequence requirements for antimicrobial activity against the human pathogen Pseudomonas aeruginosa of 277 Bac2A variants were investigated by using a luciferase-based assay. Sequence scrambling of Bac2A led to activities ranging from superior or equivalent to Bac2A to inactive, indicating that good activity was not solely dependent on the composition of amino acids or the overall charge or hydrophobicity, but rather required particular linear sequence patterns. A QSAR computational analysis was applied to analyze the data resulting in a model that supported this sequence pattern hypothesis. The activity of selected peptides was confirmed by conventional minimal inhibitory concentration (MIC) analyses with a panel of human pathogen bacteria and fungi. Circular-dichroism (CD) spectroscopy with selected peptides in liposomes and membrane depolarization assays were consistent with a relationship between structure and activity. An additional optimization process was performed involving systematic amino acid substitutions of one of the optimal scrambled peptide variants, resulting in superior active peptide variants. This process provides a cost and time effective enrichment of new candidates for drug development, increasing the chances of finding pharmacologically relevant peptides.

Amino Acid Sequence↗
PubMed2006

PAR-type thrombin receptors in renal carcinoma cells: PAR1-mediated EGFR activation promotes cell migration.

Cross-talk between G-protein-coupled receptor (GPCR) and epidermal growth factor receptor (EGFR) signaling systems is established in a wide variety of normal and neoplastic cell types. Here, we show that proteinase-activated receptor 1 (PAR1) mediates the tyrosine phosphorylation of EGFR in human renal carcinoma cells expressing PAR1 and PAR3 endogeneously. This GPCR-EGFR signal transduction pathway cross-talk requires matrix metalloproteinase activity and is involved in the regulation of renal carcinoma cell migration across a collagen barrier as shown using a Boyden chamber type assay. Our data therefore document a regulatory role of PAR1-mediated EGFR transactivation in cancer cell chemotactic migration. Further, our results underline the importance of PAR1-mediated pathways in kidney cancer cells and suggest that the thrombin/PAR1 system mediating EGFR transactivation may play a role in the progression of this tumor entity.

Cell Line, Tumor↗
PubMed2005

Solution structure of the human immunodeficiency virus type 1 p6 protein.

The human immunodeficiency virus type 1 p6 protein represents a docking site for several cellular and viral binding factors and fulfills major roles in the formation of infectious viruses. To date, however, the structure of this 52-amino acid protein, by far the smallest lentiviral protein known, either in its mature form as free p6 or as the C-terminal part of the Pr55 Gag polyprotein has not been unraveled. We have explored the high resolution structure and folding of p6 by CD and NMR spectroscopy. Under membranous solution conditions, p6 can adopt a helix-flexible helix structure; a short helix-1 (amino acids 14-18) is connected to a pronounced helix-2 (amino acids 33-44) by a flexible hinge region. Thus, p6 can be subdivided into two distinct structural and functional domains; helix-2 perfectly defines the region that binds to the virus budding factor AIP-1/ALIX, indicating that this structure is required for interaction with the endosomal sorting complex required for transport. The PTAP motif at the N terminus, comprising the primary late assembly domain, which is crucial for interaction with another cellular budding factor, Tsg101, does not exhibit secondary structure. However, the adjacent helix-1 may play an indirect role in the specific complex formation between p6 and the binding groove in Tsg101. Moreover, binding studies by NMR demonstrate that helix-2, which also comprises the LXXLF motif required for incorporation of the human immunodeficiency virus type 1 accessory protein Vpr into budding virions, specifically interacts with the Vpr binding region, indicating that under the specific solution conditions used for structure analysis, p6 adopted a functional conformation.

Amino Acid Motifs↗
PubMed2005

Proteinase-activated receptors (PARs)--the PAR3 Neo-N-terminal peptide TFRGAP interacts with PAR1.

Thrombin activates proteinase-activated receptor (PAR)1, PAR3 and PAR4 by a unique mechanism that involves cleavage of the receptor and exposure of a new N-terminal domain acting as a tethered ligand. Synthetic peptides based on the proteolytically revealed receptor sequence can selectively activate PAR1 or PAR4 independently of receptor cleavage. However, corresponding peptides for PAR3 have not been identified thus far. Here, we demonstrate that the synthetic peptide TFRGAP representing the 1st six residues of the new amino terminus of PAR3 induced ERK activation in human A-498 carcinoma cells endogeneously expressing PAR1 and PAR3. This effect was completely abolished by single alanine substitution at positions 3, 4 and 6 in the peptide. Since the specific PAR1 antagonist RWJ 56110 completely abolished TFRGAP-induced ERK activation in A-498 cells we speculate that TFRGAP does signal MAPK via interaction with PAR1. This was underlined by experiments on PAR1-/- mouse lung fibroblasts (KOLF cells) that stably overexpress human PAR1 and PAR3, respectively. While TFRGAP was without effect on ERK activation in PAR3+ KOLF cells, it induced MAPK activation in KOLF cells transfected with PAR1. These studies provide evidence that analogues of the PAR3 tethered ligand can mediate cell signaling by interaction with PAR1-type thrombin receptors.

Alanine↗
PubMed2005

SIRT1 regulates HIV transcription via Tat deacetylation.

The human immunodeficiency virus (HIV) Tat protein is acetylated by the transcriptional coactivator p300, a necessary step in Tat-mediated transactivation. We report here that Tat is deacetylated by human sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent class III protein deacetylase in vitro and in vivo. Tat and SIRT1 coimmunoprecipitate and synergistically activate the HIV promoter. Conversely, knockdown of SIRT1 via small interfering RNAs or treatment with a novel small molecule inhibitor of the SIRT1 deacetylase activity inhibit Tat-mediated transactivation of the HIV long terminal repeat. Tat transactivation is defective in SIRT1-null mouse embryonic fibroblasts and can be rescued by expression of SIRT1. These results support a model in which cycles of Tat acetylation and deacetylation regulate HIV transcription. SIRT1 recycles Tat to its unacetylated form and acts as a transcriptional coactivator during Tat transactivation.

Acetylation↗
PubMed2005

Influenza A virus protein PB1-F2: synthesis and characterization of the biologically active full length protein and related peptides.

Recently the discovery of a novel 87 amino acid influenza A virus (IAV) protein, named PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most of the known human IAV isolates. Using optimized protocols, full length biologically active sPB1-F2 and a number of fragments have been synthesized by following either the standard elongation SPPS method or by native chemical ligation of unprotected N- and C-terminal peptide fragments at the histidine and cysteine residues located in position 41 and 42 of the native sequence, respectively. The ligation procedure afforded the most efficient synthesis of sPB1-F2 and facilitated the generation of various mutants of sPB1-F2 from pre-synthesized peptide fragments. During the synthesis of sPB1-F2, the formation of succinimide and subsequent conversion to the piperidine derivative at the aspartic acid residue in position 23 was observed. This reaction was forestalled by applying specific modifications to the SPPS protocol. The chain-elongation SPPS protocol is optimal for producing small peptides of sPB1-F2, their derivatives and precursors for a subsequent ligation protocol, while the full length protein, mutants and labelled derivatives are more conveniently and efficiently synthesized by SPPS protocols that include native chemical ligation. The molecular identity of sPB1-F2 was confirmed by peptide mapping, mass spectrometry, N-terminal sequencing, (1)H NMR spectroscopy and Western blot analysis. The latter analysis afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization, a phenomenon observed both for full length sPB1-F2 and fragments thereof, as well as for its full length viral counterpart. Our synthesis protocols open the field for multiple biological and structural studies on sPB1-F2 that, similar to the molecule expressed in an IAV context, induces apoptosis and interacts with membranes in vitro and in vivo, as shown in previous studies.

Amino Acid Sequence↗
PubMed2005

Human immunodeficiency virus type 1-induced macrophage gene expression includes the p21 gene, a target for viral regulation.

In contrast to CD4+ T cells, human immunodeficiency virus type 1 (HIV-1)-infected macrophages typically resist cell death, support viral replication, and consequently, may facilitate HIV-1 transmission. To elucidate how the virus commandeers the macrophage's intracellular machinery for its benefit, we analyzed HIV-1-infected human macrophages for virus-induced gene transcription by using multiple parameters, including cDNA expression arrays. HIV-1 infection induced the transcriptional regulation of genes associated with host defense, signal transduction, apoptosis, and the cell cycle, among which the cyclin-dependent kinase inhibitor 1A (CDKN1A/p21) gene was the most prominent. p21 mRNA and protein expression followed a bimodal pattern which was initially evident during the early stages of infection, and maximum levels occurred concomitant with active HIV-1 replication. Mechanistically, viral protein R (Vpr) independently regulates p21 expression, consistent with the reduced viral replication and lack of p21 upregulation by a Vpr-negative virus. Moreover, the treatment of macrophages with p21 antisense oligonucleotides or small interfering RNAs reduced HIV-1 infection. In addition, the synthetic triterpenoid and peroxisome proliferator-activated receptor gamma ligand, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), which is known to influence p21 expression, suppressed viral replication. These data implicate p21 as a pivotal macrophage facilitator of the viral life cycle. Moreover, regulators of p21, such as CDDO, may provide an interventional approach to modulate HIV-1 replication.

Anti-HIV Agents↗
PubMed2004

Sequence of interactions in receptor-G protein coupling.

Guanine nucleotide exchange in heterotrimeric G proteins catalyzed by G protein-coupled receptors (GPCRs) is a key event in many physiological processes. The crystal structures of the GPCR rhodopsin and two G proteins as well as binding sites on both catalytically interacting proteins are known, but the temporal sequence of events leading to nucleotide exchange remains to be elucidated. We employed time-resolved near infrared light scattering to study the order in which the Galpha and Ggamma C-terminal binding sites on the holo-G protein interact with the active state of the GPCR rhodopsin (R*) in native membranes. We investigated these key binding sites within mass-tagged peptides and G proteins and found that their binding to R* is mutually exclusive. The interaction of the holo-G protein with R* requires at least one of the lipid modifications of the G protein (i.e. myristoylation of the Galpha N terminus and/or farnesylation of the Ggamma C terminus). A holo-G protein with a high affinity Galpha C terminus shows a specific change of the reaction rate in the GDP release and GTP uptake steps of catalysis. We interpret the data by a sequential fit model where (i) the initial encounter between R* and the G protein occurs with the Gbetagamma subunit, and (ii) the Galpha C-terminal tail then interacts with R* to release bound GDP, thereby decreasing the affinity of R* for the Gbetagamma subunit. The mechanism limits the time in which both C-terminal binding sites of the G protein interact simultaneously with R* to a short lived transitory state.

Amino Acid Sequence↗
PubMed2004

Sorting competition with membrane-permeable peptides in intact epithelial cells revealed discrimination of transmembrane proteins not only at the trans-Golgi network but also at pre-Golgi stages.

Transmembrane proteins destined to the basolateral cell surface of epithelial cells contain in their cytosolic domain at least two classes of sorting signals: one class promotes exit from the endoplasmic reticulum (ER) and transport to the Golgi complex, and the other class operates at the trans-Golgi network (TGN) specifying segregation into basolateral exocytic pathways. Both kinds of addressing motifs are quite diverse among different proteins. It is unclear to what extent this feature reflects alternative decoding mechanisms or variations in motifs recognized by the same sorting factor. Here we applied a novel strategy based on permeable peptide technology and temperature-sensitive model proteins to study competition between cytosolic sorting motifs in the context of mammalian living cells. We used the transduction domain of HIV-1 Tat protein to make a membrane-permeable peptide of the cytosolic tail of GtsO45, which contains a well characterized ER exit di-acidic (DIE) motif and a tyrosine-based basolateral sorting signal (YTDI). This peptide added to the media inhibited transport of GtsO45 from both ER-to-Golgi and TGN-to-basolateral cell surface in transfected Madin-Darby canine kidney cells. Instead, it did not affect the exocytic trafficking of a GtsO45-derived chimeric protein bearing 30 juxtamembrane residues from the cytosolic domain of the epidermal growth factor receptor that contains a variant ER exit motif (ERE) and an unconventional proline-based basolateral sorting signal. These results not only proved the feasibility of competing for sorting events in intact cells but also showed that distinct plasma membrane proteins can be discriminated at pre-TGN stages, and that basolateral sorting involves different recognition elements for tyrosine-based motifs and an unconventional basolateral motif.

Amino Acid Motifs↗
PubMed2004

Hepatitis C virus mutation affects proteasomal epitope processing.

The high incidence of hepatitis C virus (HCV) persistence raises the question of how HCV interferes with host immune responses. Studying a single-source HCV outbreak, we identified an HCV mutation that impaired correct carboxyterminal cleavage of an immunodominant HLA-A2-restricted CD8 cell epitope that is frequently recognized by recovered patients. The mutation, a conservative HCV nonstructural protein 3 (NS3) tyrosine to phenylalanine substitution, was absent in 54 clones of the infectious source, but present in 15/21 (71%) HLA-A2-positive and in 11/24 (46%) HLA-A2-negative patients with chronic hepatitis C. In order to analyze whether the mutation affected the processing of the HLA-A2-restricted CD8 cell epitope, mutant and wild-type NS3 polypeptides were digested in vitro with 20S constitutive proteasomes and with immunoproteasomes. The presence of the mutation resulted in impaired carboxyterminal cleavage of the epitope. In order to analyze whether impaired epitope processing affected T cell priming in vivo, HLA-A2-transgenic mice were infected with vaccinia viruses encoding either wild-type or mutant HCV NS3. The mutant induced fewer epitope-specific, IFN-gamma;-producing and fewer tetramer(+) cells than the wild type. These data demonstrate how a conservative mutation in the flanking region of an HCV epitope impairs the induction of epitope-specific CD8(+) T cells and reveal a mechanism that may contribute to viral sequence evolution in infected patients.

Amino Acid Sequence↗
PubMed2004

PAR1-type thrombin receptor stimulates migration and matrix adhesion of human colon carcinoma cells by a PKCepsilon-dependent mechanism.

The proteinase-activated receptor1 (PAR1) was characterized as a functional receptor for thrombin in cells from different tumor entities. In colon carcinoma, its function has to be defined. In this study we demonstrate that the PAR1-selective agonist peptide TFLLRN induced activation of protein kinase C isoenzymes alpha and epsilon in human HT-29 colon carcinoma cells expressing PAR1 endogeneously. On the cellular level, TFLLRN and thrombin prompted HT-29 cell migration and matrix adhesion by a PKCepsilon-dependent mechanism as concluded because of the inhibition of PAR1-mediated effects by the PKC inhibitor bisindolylmaleimide I and the PKCepsilon translocation inhibitory peptide EAVSLKPT but not by the PKC inhibitor Gö 6976. In addition, blockade of PAR1 by RWJ 56110, a selective PAR1 antagonist, fully abolished the effect of thrombin on HT-29 cell migration and adhesion. Therefore, PAR1 seems to be the responsible receptor for thrombin-induced migration and adhesion of human colon carcinoma cells including PKCepsilon as an essential signal transducer.

Blotting, Western↗