Adenoviruses using the cancer marker EphA2 as a receptor in vitro and in vivo by genetic ligand insertion into different capsid scaffolds.

Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK). This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis.

Structure and function of the hetero-oligomeric cysteine synthase complex in plants.

Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (ΔG = -33 kcal mol(-1)) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (K(D) = 30 nm) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (K(D)) and the regulation of cysteine sensitivity of SAT within the CSC.

Characterization of Hap/BAG-1 variants as RP1 binding proteins with antiapoptotic activity.

The MAPRE protein family (EB1, RP1, EB2) represents a highly conserved group of proteins that localize preferentially to the plus end of microtubules, both in the nucleus and cytoplasm. In addition, MAPRE family members are characterized by their capability to bind to the C-terminus of the adenomatous polyposis coli (APC) protein and tubulin in order to stabilize microtubules. Apart from the interaction with APC and tubulin, no other direct binding partners are known today. Because the RP1 gene product was identified in activated T cells, we set out to search for new interacting molecules in a yeast 2-hybrid system. We isolated a cDNA variant encoding for the antiapoptotic Hap/BAG-1 protein truncated by 34 amino acids at the C-terminus. In the original Hap/BAG-1 protein, the C-terminal domain is responsible for binding to Bcl-2 and Hsp/Hsc70, which is believed to be the reason for its antiapoptotic activity. Although this putative Hap/BAG-1 variant protein showed no interaction with Bcl-2 or Hsp/Hsc70, it was perfectly able to confer resistance to apoptosis. Subcellular distribution analysis revealed that the Hap/Bag-1 variant protein localized homogenously to the cytoplasm and shuttles into the nucleus in response to stress, a process that could be blocked by RP1 protein overexpression.

Retinoblastoma susceptibility gene product pRB activates hypoxia-inducible factor-1 (HIF-1).

Hypoxia-inducible factor-1 alpha (HIF-1alpha) constitutes a regulatory subunit of HIF-1, a major transcriptional activator of genes that coordinate physiological and pathological responses towards hypoxia. In order to identify novel interaction partners of HIF-1alpha we have applied T7 phage display system and identified a domain inherent in the retinoblastoma protein (pRB). The interaction between pRB and HIF-1alpha was confirmed by in vitro experiments and in transfected cells. Thereby, an HIF-1alpha domain spanning amino acids 530-694 was mapped to be required for pRB binding. Overexpression of pRB provoked transcriptional activation of HIF-1alpha under normoxia. Furthermore, the domain of pRB identified to bind HIF-1alpha in vitro is sufficient to cause HIF-1alpha transcriptional activation with the further notion that phosphorylation deficient pRB shows stronger HIF-1alpha transactivation. Using ChIP analysis, we show that HIF-1alpha responsive elements (HREs) are precipitated using alpha-pRB antibodies. Additionally, a functional interaction between pRB and HIF-1alpha is confirmed by showing that HIF-1alpha reverses the transcription repressor function of pRB.

Transcriptional stimulation by the DNA binding protein Hap46/BAG-1M involves hsp70/hsc70 molecular chaperones.

The hsp70/hsc70-associating protein Hap46 of human origin, also called BAG-1M (Bcl-2-associated athanogene 1), has been characterized previously as a DNA binding protein, which is able to stimulate transcription. By use of in vitro assays we now show that Hap46-mediated transcriptional activation can occur from linearized as well as from supercoiled circular DNA and does not require the presence of a transcription promoter. Accordingly, we observed no preferential binding of Hap46 to overlapping DNA fragments covering the sequence of the cytomegalovirus (CMV) early promoter, thus suggesting non-specific binding. The C-terminal deletion variant Hap46DeltaC47, which is unable to associate with hsp70/hsc70 molecular chaperones, produced greatly diminished effects on transcription, indicating a significant involvement of hsp70/hsc70 chaperones but not an absolute requirement. In contrast, deletion of the acidic hexarepeat region, as in variant Hap46Delta12-62, did not disturb transcriptional stimulation. While full-length Hap46 readily formed complexes with a series of structurally unrelated transcription factors, variant Hap46DeltaC47 proved incapable of doing so. Together these data suggest that transcriptional stimulation is a major biological activity of Hap46 and point to involvement of hsp70/hsc70 molecular chaperones in transcription in concert with Hap46, thus providing a link between hsp70/hsc70 molecular chaperones and components of the transcription machinery.