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Peter Henklein

Publications and source records attributed to Peter Henklein.

34 records · Page 2Linked to original sources
PubMed2004

Tat acetylation: a regulatory switch between early and late phases in HIV transcription elongation.

The HIV transcriptional activator Tat enhances the processivity of RNA polymerase II by recruiting the CyclinT1/CDK9 complex to the TAR RNA element. In addition, Tat synergizes with the histone acetyltransferase p300 and is acetylated by p300 at a single lysine residue (K50) in the TAR RNA binding domain. We have recently reported that this post-translational modification is necessary for the interaction and transcriptional synergy of Tat with the transcriptional coactivator PCAF. We have further studied the relevance of Tat acetylation during HIV transcription and generated antibodies specific for acetylated Tat (AcTat). Microinjection of anti-AcTat antibodies inhibited Tat-mediated transactivation in cells. Similarly, the specific p300 inhibitor Lys-CoA and short inhibitory RNAs specific for p300 suppressed Tat transcriptional activity. Full-length synthetic AcTat bound to TAR RNA and CyclinT1 with high affinity, but formation of the Tat-TAR-CyclinT1 ternary complex was inhibited when K50 was acetylated. Our data collectively show that Tat acetylation by p300 defines a critical step in Tat transactivation that serves to disrupt the Tat/TAR/CyclinT1 complex and helps in recruiting PCAF to the elongating RNA polymerase II.

Acetylation↗
PubMed2003

Immunisation with a synthetic prion protein-derived peptide prolongs survival times of mice orally exposed to the scrapie agent.

Several lines of evidence suggest that immunisations may be helpful in the prophylaxis and treatment of neurodegenerative amyloidoses like Alzheimer's disease and prion infections. We used a synthetic prion protein-derived peptide (PrP105-125) and a recombinant PrP fragment (PrP90-230) as antigens for the active immunisation of mice, which were subsequently infected by dietary exposure to the scrapie agent. Immunisation with PrP105-125 prolonged the survival times significantly. In contrast, immunisation with PrP90-230 or adjuvants alone had no effect on the disease development. An epitope mapping of the antibodies raised against PrP90-230 revealed that reactivities against previously defined protective epitopes were either underrepresented or absent. These results point towards the possibility to prevent prion spread via the food chain by vaccinating humans or other species at risk to contract prion diseases.

Administration, Oral↗
PubMed2003

Structural characterization of the HIV-1 Vpr N terminus: evidence of cis/trans-proline isomerism.

The 96-residue human immunodeficiency virus (HIV) accessory protein Vpr serves manifold functions in the retroviral life cycle including augmentation of viral replication in non-dividing host cells, induction of G2 cell cycle arrest, and modulation of HIV-induced apoptosis. Using a combination of dynamic light scattering, circular dichroism, and NMR spectroscopy the N terminus of Vpr is shown to be a unique domain of the molecule that behaves differently from the C-terminal domain in terms of self-association and secondary structure folding. Interestingly, the four highly conserved proline residues in the N terminus are predicted to have a high propensity for cis/trans isomerism. Thus the high resolution structure and folding of a synthetic N-terminal peptide (Vpr1-40) and smaller fragments thereof have been investigated. 1H NMR data indicate Vpr1-40 possesses helical structure between residues 17-32, and for the first time, this helix, which is bound by proline residues, was observed even in aqueous solution devoid of any detergent supplements. In addition, NMR data revealed that all of the proline residues undergo a cis/ trans isomerism to such an extent that approximately 40% of all Vpr molecules possess at least one proline in a cis conformation. This phenomenon of cis/trans isomerism, which is unprecedented for HIV-1 Vpr, not only provides an explanation for the molecular heterogeneity observed in the full-length molecule but also indicates that in vivo the folding and function of Vpr should depend on a cis/trans-proline isomerase activity, particularly as two of the proline residues in positions 14 and 35 show considerable amounts of cis isomers. This prediction correlates well with our recent observation (Zander, K., Sherman, M. P., Tessmer, U., Bruns, K., Wray, V., Prechtel, A. T., Schubert, E., Henklein, P., Luban, J., Neidleman, J., Greene, W. C., and Schubert, U. (2003) J. Biol. Chem. 278, 43170-43181) of a functional interaction between the major cellular isomerase cyclophilin A and Vpr, both of which are incorporated into HIV-1 virions.

Amino Acid Sequence↗
PubMed2003

Cyclophilin A interacts with HIV-1 Vpr and is required for its functional expression.

Viral protein R (Vpr) of human immunodeficiency virus, type 1 (HIV-1) is the major virion-associated accessory protein that affects a number of biological functions in the retroviral life cycle, including promotion of the transport of the preintegration complex into the nucleus and the induction of G2 host cell cycle arrest. Our recent investigation of the conformational heterogeneity of the proline residues in the N terminus of Vpr suggested a functional interaction between Vpr and a host peptidylprolyl cis/trans isomerase (PPIase) that might regulate the cis/trans interconversion of the imidic bond within the conserved proline residues of Vpr in vivo. Using surface plasmon resonance spectroscopy, Far Western blot, and pulldown experiments a physical interaction of Vpr with the major host PPIase cyclophilin A (CypA) is now demonstrated. The interaction domain involves the N-terminal region of Vpr including an essential role for proline in position 35. The CypA inhibitor cyclosporin A and non-immunosuppressive PPIase inhibitors such as NIM811 and sanglifehrin A block expression of Vpr without affecting pre- or post-translational events such as transcription, intracellular transport, or virus incorporation of Vpr. Similarly to CypA inhibition, Vpr expression is also reduced in HIV-1 infected CypA-/- knock-out T cells. This study thus shows that in addition to the interaction between CypA and HIV-1 capsid occurring during early steps in virus replication, CypA is also important for the de novo synthesis of Vpr and that in the absence of CypA activity, the Vpr-mediated cell cycle arrest is completely lost in HIV-1-infected T cells.

Amino Acid Sequence↗
PubMed2003

Complex polyfluoride additives in Fmoc-amino acid fluoride coupling processes. Enhanced reactivity and avoidance of stereomutation.

[reaction: see text] Isolated Fmoc amino acid fluorides have previously been shown to be among the most efficient reagents for peptide bond formation. Now, it has been found that anionic, polyhydrogen fluoride additives are capable of diverting many of the classical peptide coupling processes to acid fluoride couplings. Examples include the use of N-HBTU or N-HATU and the carbodiimide technique. As HF-containing species, these additives provide a more suitable medium for the coupling of systems that are sensitive to loss of configuration at the reactive carboxyl function.

Amino Acid Sequence↗
PubMed2003

Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome.

The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphoryl ates proteins such as c-Jun and p53 with consequence for their ubiquitin (Ub)-dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (PKD) co-purify with CSN. Immunoprecipitation and far-western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold-labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co-immuno precipitates with CSN from HeLa cells. CK2 binds to DeltaCSN3(111-403) and CSN7, whereas PKD interacts with full-length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c-Jun as well as p53. CK2 phosphoryl ates Thr155, which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN-associated kinases and induce degradation of c-Jun in HeLa cells. Curcumin treatment results in elevated amounts of c-Jun-Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation.

Adenosine Triphosphate↗
PubMed2003

An essential role for tripeptidyl peptidase in the generation of an MHC class I epitope.

Most of the peptides presented by major histocompatibility complex (MHC) class I molecules require processing by proteasomes. Tripeptidyl peptidase II (TPPII), an aminopeptidase with endoproteolytic activity, may also have a role in antigen processing. Here, we analyzed the processing and presentation of the immunodominant human immunodeficiency virus epitope HIV-Nef(73-82) in human dendritic cells. We found that inhibition of proteasome activity did not impair Nef(73-82) epitope presentation. In contrast, specific inhibition of TPPII led to a reduction of Nef(73-82) epitope presentation. We propose that TPPII can act in combination with or independent of the proteasome system and can generate epitopes that evade generation by the proteasome-system.

Aminopeptidases↗
PubMed2003

Identification and characterization of SmD183-119-reactive T cells that provide T cell help for pathogenic anti-double-stranded DNA antibodies.

OBJECTIVE: The C-terminal peptide of amino acids 83-119 of the SmD1 protein is a target of the autoimmune response in human and murine lupus. This study was undertaken to test the hypothesis that SmD1(83-119)-reactive T cells play a crucial role in the generation of pathogenic anti-double-stranded DNA (anti-dsDNA) antibodies. METHODS: Splenic or lymph node T cells derived from unmanipulated as well as SmD1(83-119)-immunized NZB/NZW mice were analyzed in vitro by enzyme-linked immunospot (ELISpot) assay to determine T cell help for anti-dsDNA generation induced by the SmD1(83-119) peptide. Cytokines expressed by these T cells were measured by ELISpot assay, enzyme-linked immunosorbent assay, and flow cytometry. SmD1(83-119)- and ovalbumin-specific T cell lines were generated and characterized. RESULTS: The SmD1(83-119) peptide, but not the control peptides, significantly increased the in vitro generation of anti-dsDNA antibodies in cultures from unmanipulated NZB/NZW mice. Interferon-gamma (IFNgamma), interleukin-2 (IL-2), IL-4, transforming growth factor beta, and IL-10 production increased in response to the peptide in young mice; only IFNgamma and IL-2 were increased in older, diseased mice. Activation of SmD1(83-119)-reactive T cells by immunization of NZB/NZW mice resulted in elevated anti-dsDNA synthesis and, later, increased antibodies to SmD1(83-119). Most cells in SmD1(83-119)-specific CD4+ T cell lines helping both antibodies had increased intracellular expression of IFNgamma, and most expressed both IFNgamma and IL-4. CONCLUSION: The SmD1(83-119) peptide plays an important role in generating T cell help for autoantibodies, including anti-dsDNA, and activates different subsets of T cells as defined by distinct cytokine expression. This peptide is an interesting target structure for the modulation of autoreactive T cells, and its characterization may contribute to our understanding of the role of autoantigen-reactive T cells in the pathogenesis of SLE.

Animals↗
PubMed2003

Production, purification, and characterization of rat pro-CCK from serum-free adapted Drosophila cells.

The precursor of cholecystokinin (pro-CCK) was expressed and purified from media of stably transfected D.Mel-2 cell as an V5-His tagged fusion protein. Its identity was confirmed using SDS-PAGE, immunoblotting, gel filtration chromatography, HPLC, and Mass Spectroscopy. Two major forms of pro-CCK were found with a molecular weight of about 14.4 and 11.3 kDa. The smaller form represents the V5-His tagged pro-CCK after cleavage at a single arginine residue at CCK-58. This cleavage is probably being performed by endogenous proteases in these cells. Purification of the desired larger form of pro-CCK is possible using a nickel column with a recovery of about 20%, yielding 500 microg/L media. The purified protein is stable for several months and can be used for further functional studies of pro-CCK.

Amino Acid Sequence↗
PubMed2003

Acetylation of Tat defines a cyclinT1-independent step in HIV transactivation.

The HIV transcriptional activator Tat is acetylated by p300 at a single lysine residue in the TAR RNA binding domain. We have generated monoclonal and polyclonal antibodies specific for the acetylated form of Tat (AcTat). Microinjection of anti-AcTat antibodies inhibited Tat-mediated transactivation in cells. Similarly, the p300 inhibitor Lys-CoA and siRNA specific for p300 suppressed Tat transcriptional activity. Full-length synthetic AcTat bound to TAR RNA with the same affinity as unacetylated Tat, but formation of a Tat-TAR-CyclinT1 ternary complex was completely inhibited in the presence of AcTat. We propose that Tat acetylation may help in dissociating the Tat cofactor CyclinT1 from TAR RNA and serve to transfer Tat onto the elongating RNA polymerase II.

Acetylation↗
PubMed2003

Hepatitis B virus HBx peptide 116-138 and proteasome activator PA28 compete for binding to the proteasome alpha4/MC6 subunit.

PA28 is a modulator of the 20S proteasome. The PA28 binding sites on the 20S proteasome are still not well defined. Using yeast two-hybrid interaction assays and proteasome inactivation kinetics we provide evidence that the proteasome alpha4 subunit is one of the PA28 binding sites. This finding is supported by the observation that a hepatitis B virus X protein-derived polypeptide habouring the alpha4 proteasome subunit binding motif impairs the activation of 20S proteasomes by PA28.

Algorithms↗
PubMed2002

The RTP site shared by the HIV-1 Tat protein and the 11S regulator subunit alpha is crucial for their effects on proteasome function including antigen processing.

The human immunodeficiency virus-1 Tat protein inhibits the peptidase activity of the 20S proteasome and competes with the 11S regulator/PA28 for binding to the 20S proteasome. Structural comparison revealed a common site in the Tat protein and the 11S regulator alpha-subunit (REGalpha) called the REG/Tat-proteasome-binding (RTP) site. Kinetic assays found amino acid residues Lys51, Arg52 and Asp67 forming the RTP site of Tat to be responsible for the effects on proteasomes in vitro. The RTP site identified in REGalpha consists of the residues Glu235, Lys236 and Lys239. Mutation of the REGalpha amino acid residues Glu235 and Lys236 to Ala resulted in an REGalpha mutant that lost the ability to activate the 20S proteasome even though it still forms complexes with REGbeta and binds to the 20S proteasome. The REGalpha RTP site is needed to enhance the presentation of a cytomegalovirus pp89 protein-derived epitope by MHC class I molecules in mouse fibroblasts. Cell experiments demonstrate that the Tat amino acid residues 37-72 are necessary for the interaction of the viral protein with proteasomes in vivo. Full-length Tat and the Tat peptide 37-72 suppressed 11S regulator-mediated presentation of the pp89 epitope. In contrast, the Tat peptide 37-72 with mutations of amino acid residues Lys51, Arg52 and Asp67 to Ala was not able to reduce antigen presentation.

Amino Acid Sequence↗
PubMed2002

HIV-1 Vpr displays natural protein-transducing properties: implications for viral pathogenesis.

The 14-kDa Vpr protein of human immunodeficiency virus type 1 (HIV-1) serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest in proliferating T lymphocytes, and the modulation of HIV-1-induced apoptosis. Extracellular Vpr has been detected in the sera and cerebral spinal fluid of HIV-infected patients. However, it is not known whether such forms of Vpr are biologically active. Vpr contains a carboxy-terminal basic amino acid rich segment stretch that is homologous to domains that mediate the energy- and receptor-independent cellular uptake of polypeptides by a process termed protein transduction. Similar functional protein-transducing domains are present in HIV-1 Tat, herpes simplex virus-1 DNA-binding protein VP22, and the Drosophila antennapedia homeotic transcription factor. We now demonstrate effective transduction of biologically active, synthetic Vpr (sVpr) as well as the Vpr-beta-galactosidase fusion protein. However, in contrast to other transducing proteins, Vpr transduction is not enhanced by protein denaturation, and Vpr's carboxy-terminal basic domain alone is not sufficient for its transduction across biological membranes. In contrast, the full-length Vpr protein effectively transduces a broad array of cells, leading to dose-dependent G2 cell-cycle arrest and apoptosis. Addition of Vpr into the extracellular medium also rescues the replication of Vpr-deficient strains of HIV-1 in human macrophage cultures. Native Vpr may thus be optimized for protein transduction, a feature that might enhance and extend the pathological effects of HIV infection.

Cell Line, Transformed↗
PubMed2002

Transcriptional synergy between Tat and PCAF is dependent on the binding of acetylated Tat to the PCAF bromodomain.

The human immunodeficiency virus (HIV) Tat protein plays an essential role in promoting efficient transcriptional elongation of viral transcripts. We report that the transcriptional co-activator PCAF and Tat interact and synergize to activate the HIV promoter. The binding of Tat and PCAF in vitro and in vivo is dependent on the acetylated state of Lys50 of Tat and on the PCAF bromodomain. Structural analysis of the acetylated Tat peptide bound to the PCAF bromodomain defined amino acids Y47 and R53 in Tat and V763, Y802, and Y809 in PCAF as critical interaction points between the two proteins. Mutation of each of these residues in either Tat or PCAF inhibited in a cumulative manner the Tat-PCAF interaction in vitro and in vivo, and abrogated the synergistic activation of the HIV promoter by both proteins. These observations demonstrate that acetylation of Tat establishes a novel protein-protein interaction domain at the surface of Tat that is necessary for the transcriptional activation of the HIV promoter.

Acetyltransferases↗
PubMed1996

Specificity of Chicken (IgY) versus Rabbit (IgG) Antibodies Raised against Cholecystokinin Octapeptide (CCK-8).

Antibodies produced in chickens (egg yolk Antibody-IgY) and rabbits against CCK-8 TyrSE (a C-terminal extended CCK-version) were compared with respect to their specificity against several modified CCK-sequences by means of radioimmunoassay and spot blot assay. The content of neuronal CCK was determined by using both an "avian" and a "mammalian" RIA. The IC50 values obtained indicate differences between the binding capacity of rabbit and chicken Antibody, respectively. Supported by the data from spot blot assay, it appears, that the avian Antibody binding activity was directed primarily towards short CCK-sequences whereas the longer sequences are less well recognised in contrast to the mammalian antibody. Probably, these differences may be due to characteristics regarding the shape of the molecules (caused also by fixation processes necessary for blotting procedures) as well as to structural differences between avian and mammalian antibodies itself (both antibodies originate from quite different immune systems). By comparing the quantitative CCK data (avian versus mammalian RIA) a significant correlation could be observed. Immunohistochemical studies using avian antibodies revealed a neuronal CCK pattern different from those using rabbit antibodies. These results are discussed on the basis of the specificity studies.

Journal Article↗