Jochen Heukeshoven

A Novel Human p53 Isoform Is an Essential Element of the ATR-Intra-S Phase Checkpoint

The archetypal human tumor suppressor p53 is considered to have unique transactivation properties. The assumption is based on the fact that additionally identified human p53 isoforms lack transcriptional activity. However, we provide evidence for the existence of an alternatively spliced p53 isoform (Deltap53) that exerts its transcriptional activity independent from p53. In contrast to p53, Deltap53 transactivates the endogenous p21 and 14-3-3sigma but not the mdm2, bax, and PIG3 promoter. Cell cycle studies showed that Deltap53 displays its differential transcriptional activity only in damaged S phase cells. Upon activation of the ATR-intra-S phase checkpoint, Deltap53, but not p53, transactivates the Cdk inhibitor p21. Induction of p21 results in downregulation of cyclin A-Cdk activity and accordingly attenuation of S phase progression. Data demonstrate that the Deltap53-p21-cyclin A-Cdk pathway is crucial to facilitate uncoupling of repair and replication events, indicating that Deltap53 is an essential element of the ATR-intra-S phase checkpoint.

Identification of cellular deoxyhypusine synthase as a novel target for antiretroviral therapy

The introduction of highly active antiretroviral therapy (HAART) has significantly decreased morbidity and mortality among patients infected with HIV-1. However, HIV-1 can acquire resistance against all currently available antiretroviral drugs targeting viral reverse transcriptase, protease, and gp41. Moreover, in a growing number of patients, the development of multidrug-resistant viruses compromises HAART efficacy and limits therapeutic options. Therefore, it is an ongoing task to develop new drugs and to identify new targets for antiretroviral therapy. Here, we identified the guanylhydrazone CNI-1493 as an efficient inhibitor of human deoxyhypusine synthase (DHS). By inhibiting DHS, this compound suppresses hypusine formation and, thereby, activation of eukaryotic initiation factor 5A (eIF-5A), a cellular cofactor of the HIV-1 Rev regulatory protein. We demonstrate that inhibition of DHS by CNI-1493 or RNA interference efficiently suppressed the retroviral replication cycle in cell culture and primary cells. We show that CNI-1493 inhibits replication of macrophage- and T cell-tropic laboratory strains, clinical isolates, and viral strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Moreover, no measurable drug-induced adverse effects on cell cycle transition, apoptosis, and general cytotoxicity were observed. Therefore, human DHS represents a novel and promising drug target for the development of advanced antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.

Interaction of casein kinase 1 delta (CK1δ) with post-Golgi structures, microtubules and the spindle apparatus

Members of the casein kinase 1 family of serine/threonine kinases are highly conserved from yeast to mammals and seem to play an important role in vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. We here report that in interphase cells of various mammalian species casein kinase 1 delta (CK1delta) specifically interacts with the trans Golgi network and cytoplasmic, granular particles that associate with microtubules. Furthermore, at mitosis CK1delta is recruited to the spindle apparatus and the centrosomes in cells, which have been exposed to DNA-damaging agents like etoposide or gammairradiation. In addition, determination of the affinity of CK1delta to different tubulin isoforms in immunoprecipitation-Western analysis revealed a dramatically enhanced complex formation between CK1delta and tubulins from mitotic extracts after introducing DNA damage. The high affinity of CK1delta to the spindle apparatus in DNA-damaged cells and its ability to phosphorylate several microtubule-associated proteins points to a regulatory role of CK1delta at mitosis.

Reversed-phase high-performance liquid chromatography of virus proteins and other large hydrophobic proteins in formic acid containing solvents

The excellent dissolving capacity of formic acid together with a propanol-2 gradient is utilized in a new system for reversed-phase high-performance liquid chromatographic separation of poliovirus polypeptides and a variety of large proteins. Differences in elution characteristics were detected between reduced and non-reduced proteins containing disulphide bridges as well as proteins modified at cysteinyl residues. The retention coefficients of single amino acids were used to calculate those of proteins. The correlation of calculated coefficients with actual retention times indicates that some proteins are bound via their full, unfolded length to the reversed-phase support, whereas others partly preserved their secondary structure. Treatment of proteins with sodium dodecyl sulphate prior to injection dissociates these structural elements and leads to an increase in retention times. The high resolution of the system described should be applicable to the isolation and characterization of components of mixtures of proteins, particularly those of water-insoluble proteins of membranes or viruses, on the analytical and semi-preparative scales.

Characterization of a solvent system for separation of water-insoluble poliovirus proteins by reversed-phase high-performance liquid chromatography

Formic acid in high concentration is an extremely potent solvent for proteins, particularly for hydrophobic ones. 60% Formic acid, necessary for solubilization of structural polypeptides of poliovirus and other proteins, modified at the cysteines, was used together with 2-propanol or acetonitrile as organic modifier for gradient elution in reversed-phase high-performance liquid chromatography. Several reversed-phase columns were tested. In each case, polypeptides were eluted quantitatively. It was demonstrated that this solvent system, with its high proportion of formic acid, did not affect the size, hydrophobicity and charge of the separated polypeptides. By injection into rabbits of poliovirus polypeptides, obtained in high purity by chromatography in the new solvent system, monospecific antibodies were induced, the specificity of which was determined by immunoprecipitation.

Analysis of Nucleocytoplasmic Trafficking of the HuR Ligand APRIL and Its Influence on CD83 Expression

Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system and are able to sensitize even naïve T cells. Mature DC are characterized by expression of CD83, a surface molecule that is proposed to be involved in efficient T cell activation. It has been recently shown that CD83 mRNA is transported from the nucleus to the cytoplasm in a HuR- and CRM1-dependent manner. Therefore we here investigated the impact of two known protein ligands of HuR, pp32 and APRIL, on CD83 expression. Both pp32 (ANP32A) and APRIL (ANP32B) are shuttle proteins, and it has been reported earlier that these HuR ligands can act as adaptors that link HuR and the CRM1-specific nuclear export pathway. By employing RNA interference (RNAi) technology we demonstrate that pp32 is dispensable for CD83 expression, whereas APRIL contributes to the nuclear export and subsequent translation of CD83 mRNA. Furthermore, we have determined the nuclear import signal (NLS) as well as the nuclear export signal (NES) of human APRIL. Moreover, we analyzed the status of phosphorylation of endogenous APRIL and identified threonine 244 to be an as yet unrecognized phosphate acceptor. Finally, we were able to show that phosphorylation of this specific amino acid residue regulates the nuclear export of APRIL. In sum, we report here the signal sequences in APRIL that mediate its intracellular trafficking and provide evidence that this protein ligand of HuR is an important player in the post-transcriptional regulation of CD83 expression by affecting the nucleocytoplasmic translocation of CD83 mRNA.

Studies on the glycosylation of n acetylneuraminic acid

Abstract Glycal derivatives of N-acetylneuraminic acid were prepared and their N-iodosuccinimide-mediated glycosylation shown to proceed only with most reactive alcohols, Their reduced enol ether reactivity is attributed to the α unsaturated ester reature. Thus several reduced glycal derivatives were synthesized. These couid be glycosyiatec with simple alcohols as well as otner saccnarides as aglycones in averaae to modest yields with the trans-diaxiai additions compounas prevailing. A number or selective and specific prepparation led to both the anomeric phenylthioglycosides or N dcety Ineuraminic acid. These could be used in phenylmercurl triflate-promoted glycosylations to afford several derivatives.

Induction of neutralizing antibodies by all three structural poliovirus polypeptides

Structural polypeptides VP1, VP2, and VP3 of poliovirus, type 1, strain Mahoney, induced low, but clearly demonstrable levels of neutralizing antibodies in rabbits. Half-neutralization titers of antisera for 1000 PFU/ml of poliovirus were in the range of 0.5 to 1.5 log10 units. Polypeptides were isolated by three independent procedures including different dissociation and separation methods to circumvent their denaturation and possible contamination by neighboring fractions. The best and fastest method was dissociation of poliovirus by formic acid and separation of polypeptides by reverse-phase high-performance liquid chromatography.

Rapid analytical and preparative separation of structural polypeptides of poliovirus by reverse-phase high-performance liquid chromatography

A reverse-phase high-performance liquid-chromatography (RP-HPLC) technique was developed for rapid (20 min) and effective separation of the structural polypeptides of poliovirus of strains of all three serological types. The method is useful for quantitative analysis as well as for isolation of polypeptides on a micropreparative scale for chemical, biochemical and immunological studies. All four virus polypeptides (VP1 to VP4) were obtained quantitatively in high purity by this one-step procedure. The solvents used were volatile and easily removed by evaporation, giving dry, amorphous polypeptides free of any additives. The separation mechanism of this RP-HPLC is based on the hydrophobicity of the protein surface. It is distinct from other separation methods such as molecular sieving and isoelectric focusing. Therefore, differences in primary and secondary structure of a polypeptide can be detected by RP-HPLC.

Average and maximum charge states of arginine- containing dendrimer-like peptide ions formed by electrospray ionization

The maximum and average charge states formed by electrospray ionization of dendrimer-like multiple antigenic peptides (MAPs) which differ in structure only in the presence of an arginine residue at the N-termini of their four peptide chains have been investigated. Stepwise addition of arginine residues leads to increased charging. It has been found that the average charge state is linearly correlated to the number of arginine residues which allows the conclusion that the four peptide chains are effectively independent. The average charge state z av is shifted with each added arginine residue by roughly 0.3 units towards lower m/z ratios. Modification of the alpha-amino groups by acetylation reduces z av as compared with the corresponding non-modified model peptides. This suggests that the N-terminal arginineis to some extent protonatedon both its alpha-amino group and its side-chain guanidino group. The Coulomb repulsion is presumably reduced through intramolecular charge solvation in the N-terminal part of the peptide chains.