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Ursula Dietrich

Publications and source records attributed to Ursula Dietrich.

8 recordsLinked to original sources
PubMed2006

A tryptophan-rich hexapeptide inhibits nucleic acid destabilization chaperoned by the HIV-1 nucleocapsid protein.

The nucleocapsid protein (NC) of HIV-1 exerts critical functions in viral genome replication and virus assembly. Since the recognition of target nucleic acids is required in the initial step of most NC-mediated processes, attempts were made to find small molecules capable of competing with this recognition. In particular, several Trp-rich hexapeptides were recently found to strongly bind RNA sequences targeted by NC. To further validate these peptides as potential anti-NC agents, we studied the ability of Ac-HKWPWW-NH2, taken as a representative, to interfere with the NC chaperone properties required during reverse transcription. Using NMR and steady-state and time-resolved fluorescence spectroscopy, we characterized the structure of Ac-HKWPWW-NH2 as well as its binding to viral sequences such as TAR and PBS involved in the two obligatory strand transfers of reverse transcription. Results show that Ac-HKWPWW-NH2 exhibits an almost symmetric cis-trans equilibrium at the level of the Pro residue where it is structured. The peptide binds both TAR and PBS sequences with low micromolar affinities. The cis-Pro and trans-Pro conformations of the peptide bind with comparable affinities to (-)PBS, mainly through stacking interactions between the Trp residues and the (-)PBS bases. Though all three Trp residues may contribute to the (-)PBS/Ac-HKWPWW-NH2 complex formation, Trp3 and Trp5 residues are the key residues in the complexes with the cis-Pro and trans-Pro conformations, respectively. Moreover, Ac-HKWPWW-NH2 stabilizes cTAR secondary structure and largely inhibits the NC-directed melting of cTAR. This further strengthens the interest of this peptide for deriving modified peptides capable of inhibiting NC and HIV-1 replication.

Amino Acid Sequence↗
PubMed2006

Stable expression of soluble therapeutic peptides in eukaryotic cells by multimerisation: application to the HIV-1 fusion inhibitory peptide C46.

A major drawback of therapeutic peptides is their short half-life, which results in the need for multiple applications and high synthesis costs. To overcome this, we established a eukaryotic expression system that allows the stable expression of small therapeutic peptides by multimerisation. By inserting the sequence encoding the therapeutic peptide between a signal peptide and the multimerising domain of the alpha-chain from the human C4bp plasma protein, therapeutic peptides as small as 5 kDa are secreted as multimers from transfected cells; this allows easy purification. As proof of principle, we show that the T20-derived HIV-1 fusion inhibitory peptide C46 in its multimeric form: i) was efficiently secreted, ii) was more stable than the current antiviral drug T20 in vitro and in vivo, and iii) inihibited HIV-1 entry with similar efficiency in vitro. Besides the gain in stability, multimerisation also leads to increased valency and allows the combination of several therapeutic functions. Furthermore, by expressing the multimers from cells, post-translational modifications could easily be introduced.

Amino Acid Sequence↗
PubMed2005

A peptide inhibitor of HIV-1 assembly in vitro.

Formation of infectious HIV-1 involves assembly of Gag polyproteins into immature particles and subsequent assembly of mature capsids after proteolytic disassembly of the Gag shell. We report a 12-mer peptide, capsid assembly inhibitor (CAI), that binds the capsid (CA) domain of Gag and inhibits assembly of immature- and mature-like capsid particles in vitro. CAI was identified by phage display screening among a group of peptides with similar sequences that bind to a single reactive site in CA. Its binding site was mapped to CA residues 169-191, with an additional contribution from the last helix of CA. This result was confirmed by a separate X-ray structure analysis showing that CAI inserts into a conserved hydrophobic groove and alters the CA dimer interface. The CAI binding site is a new target for antiviral development, and CAI is the first known inhibitor directed against assembly of immature HIV-1.

Amino Acid Sequence↗
PubMed2005

3D-Epitope-Explorer (3DEX): localization of conformational epitopes within three-dimensional structures of proteins.

Neutralizing antibodies often recognize conformational, discontinuous epitopes. Linear peptides mimicking such conformational epitopes can be selected from phage display peptide libraries by screening with the respective antibodies. However, it is difficult to localize these "mimotopes" within the three-dimensional (3D) structures of the target proteins. Knowledge of conformational epitopes of neutralizing antibodies would help to design antigens able to elicit protective immune responses. Therefore, we provide here a software that allows to localize linear peptide sequences within 3D structures of proteins. The 3D-Epitope-Explorer (3DEX) software allows to map conformational epitopes in 3D protein structures based on an algorithm that takes into account the physicochemical neighborhood of C(alpha)- or C(beta)-atoms of individual amino acids. A given amino acid of a peptide sequence is localized within the protein and the software searches within predefined distances for the amino acids neighboring that amino acid in the peptide. Surface exposure of the amino acids can also be taken into consideration. The procedure is then repeated for the remaining amino acids of the peptide. The introduction of a joker function allows to map peptide mimotopes, which do not necessarily have 100% sequence homology to the protein. Using this software we were able to localize mimotopes selected from phage displayed peptide libraries with polyclonal antibodies from HIV-positive patient plasma within the 3D structure of gp120, the exterior glycoprotein of HIV-1. We also analyzed two recently published peptide sequences corresponding to known conformational epitopes to further confirm the integrity of 3DEX.

Algorithms↗
PubMed2005

Requirement of multiple phage displayed peptide libraries for optimal mapping of a conformational antibody epitope on CCR5.

In the absence of information from crystallography, conformational epitopes can often be discerned by antibody screening of phage displayed random peptide libraries. However the context in which the peptide is displayed, and the number of copies displayed in the library, can influence results and interpretations. Here, the monoclonal antibodies 3A9 specific for the transmembrane chemokine receptor CCR5, and CII-C1 specific for type II collagen, were used to screen multiple phage-displayed peptide libraries in which peptides were displayed in either the pIII or pVIII coat proteins. ELISA was used to test for reactivity and cross-inhibitory activity of isolated phage clones. Based on sequences of reactive phage inserts, epitope motifs were initially inferred from a molecular model of CCR5 and subsequently confirmed experimentally using mutagenesis to alanine. For each mAb, phage sequences from pIII biopannings were more diverse than from pVIII biopannings. Notably, sequences from either biopanning were cross-inhibitory despite a lack of linear sequence homology. For CCR5, residues 88H and 94W in the first loop of CCR5 were identified by pIII biopannings, and 7S9IYD11 at the N-terminus by pVIII biopannings. Thus conformational epitopes can be identified using phage display, but optimal mapping of complex epitopes can require the use of multiple peptide libraries.

Amino Acid Motifs↗
PubMed2005

Feline glaucomas.

Cats are usually presented at a very late stage in the course of glaucoma when the eye is already blind. Secondary glaucoma because of another underlying ocular lesion is the most common form of glaucoma in the cat and is frequently associated with chronic anterior uveitis or intraocular neoplasia. Chronic stages of glaucoma in the cat are characterized by buphthalmus, anterior lens luxation, and exposure keratitis secondary to the enlarged globe. Ophthalmoscopic signs of glaucomatous retinal degeneration are only noticed in very advanced stages. Treatment of glaucoma in cats is usually aimed to keep the eye comfortable and within a normal intraocular pressure range. However, many antiglaucoma medications that are successfully used in humans and dogs are not very well tolerated by cats and, therefore, the selection of recommended drugs is limited in this species.

Animals↗
PubMedDate not supplied

The role of neutralizing antibodies in HIV infection.

Neutralizing antibodies against HIV-1 play an important role in preventing viral infections. Less clear is their role in the containment of viral replication in infected individuals. However, evidence is accumulating that neutralizing antibodies may help the cellular arm of the immune response to prevent or delay the progression to AIDS. Detection of neutralizing antibodies depends on the in vitro neutralization assays used, and standardization of the assays is essential in order to be able to compare the magnitude and quality of a neutralizing antibody response in sera or other fluids from HIV-infected patients, uninfected HIV-1 exposed persons, or vaccinated animals/persons. Viral mechanisms to prevent neutralization include high variability and extensive glycosylation of the Envelope proteins, Envelope trimerization and shedding as well as late exposure of functionally important entry domains by conformational changes induced upon CD4 binding. These are also the difficulties encountered in the design of immunogens able to induce neutralizing antibodies upon vaccination.

AIDS Vaccines↗