hADA3 is required for p53 activity.

The tumor suppressor protein p53 is a transcription factor that is frequently mutated in human cancers. In response to DNA damage, p53 protein is stabilized and activated by post-translational modifications that enable it to induce either apoptosis or cell cycle arrest. Using a novel yeast p53 dissociator assay, we identify hADA3, a part of histone acetyltransferase complexes, as an important cofactor for p53 activity. p53 and hADA3 physically interact in human cells. This interaction is enhanced dramatically after DNA damage due to phosphorylation event(s) in the p53 N-terminus. Proper hADA3 function is essential for full transcriptional activity of p53 and p53-mediated apoptosis.

Genetic selection of intragenic suppressor mutations that reverse the effect of common p53 cancer mutations.

Several lines of evidence suggest that the presence of the wild-type tumor suppressor gene p53 in human cancers correlates well with successful anti-cancer therapy. Restoration of wild-type p53 function to cancer cells that have lost it might therefore improve treatment outcomes. Using a systematic yeast genetic approach, we selected second-site suppressor mutations that can overcome the deleterious effects of common p53 cancer mutations in human cells. We identified several suppressor mutations for the V143A, G245S and R249S cancer mutations. The beneficial effects of these suppressor mutations were demonstrated using mammalian reporter gene and apoptosis assays. Further experiments showed that these suppressor mutations could override additional p53 cancer mutations. The mechanisms of such suppressor mutations can be elucidated by structural studies, ultimately leading to a framework for the discovery of small molecules able to stabilize p53 mutants.

Tag games in yeast: the two-hybrid system and beyond.

The yeast Saccharomyces cerevisiae and the one- and two-hybrid systems are essential genetic tools for studying the macromolecular interactions that define all living organisms. Newly developed variations on this theme can now address an even bigger set of questions. Reverse one- and two-hybrid systems can identify factors that dissociate or abrogate defined macromolecular interactions. Different forms of three-hybrid systems can evaluate the complex interplay of proteins with RNAs, peptide ligands, small organic ligands or protein kinases. Finally, the ubiquitin-based split-protein sensor and the Sos recruitment systems promise to overcome some limitations of conventional two-hybrid systems.

Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions.

Macromolecular interactions define many biological phenomena. Although genetic methods are available to identify novel protein-protein and DNA-protein interactions, no genetic system has thus far been described to identify molecules or mutations that dissociate known interactions. Herein, we describe genetic systems that detect such events in the yeast Saccharomyces cerevisiae. We have engineered yeast strains in which the interaction of two proteins expressed in the context of the two-hybrid system or the interaction between a DNA-binding protein and its binding site in the context of the one-hybrid system is deleterious to growth. Under these conditions, dissociation of the interaction provides a selective growth advantage, thereby facilitating detection. These methods referred to as the "reverse two-hybrid system" and "reverse one-hybrid system" facilitate the study of the structure-function relationships and regulation of protein-protein and DNA-protein interactions. They should also facilitate the selection of dissociator molecules that could be used as therapeutic agents.

Dominant-negative p53 mutations selected in yeast hit cancer hot spots.

Clinically important mutant p53 proteins may be tumorigenic through a dominant-negative mechanism or due to a gain-of-function. Examples for both hypotheses have been described; however, it remains unclear to what extent they apply to TP53 mutations in general. Here it is shown that the mutational spectrum of dominant-negative p53 mutants selected in a novel yeast assay correlates tightly with p53 mutations in cancer. Two classes of dominant-negative mutations are described; the more dominant one affects codons that are essential for the stabilization of the DNA-binding surface of the p53 core domain and for the direct interaction of p53 with its DNA binding sites. These results predict that the vast majority of TP53 mutations leading to cancer do so in a dominant-negative fashion.

Hydroxybenzoyl-CoA reductase: coupling kinetics and electrochemistry to derive enzyme mechanisms.

Hydroxybenzoyl-CoA reductase (HBCR) is an iron-sulfur protein that is involved in the metabolism of aromatic compounds. It catalyzes the two-electron reduction of hydroxybenzoyl-CoA to benzoyl-CoA. In the work described here, kinetic schemes were derived for HBCR and for several classes of redox enzymes and redox-activated enzymes. Introduction of the Nernst equation into the rate laws led to the development of novel relationships between the ambient redox potential, the midpoint potential of the enzyme active site, and the kinetic parameter, V/K. By coupling electrochemistry and steady-state kinetics, mechanistic information could be obtained that could not be determined by either method alone. For HBCR, the relationship between the kinetic parameter V/K and the ambient electrochemical potential of the assay mixture was found to be: apparent V/Km = Vmax/(Km(1 + exp[nF/RT(E - E(o)e)])), where n is the number of electrons involved in the redox process, F is the Faraday constant, R is the gas constant, T is the temperature in K, E is the applied potential, and E(o)e is the redox potential of a redox-active catalytic site on the enzyme. Coupling kinetics with electrochemistry yielded the E(o)e (-350 mV vs NHE) for HBCR and maximum values under optimal redox conditions for kcat and kcat/Km (9 s-1 and 1.8 x 10(5) M-1 s-1, respectively). In addition, theory was developed that could distinguish a single two-electron transfer mechanism from one involving two successive one-electron transfers. HBCR was found to be in the latter class. Interestingly, the derived mechanism for HBCR is similar to that of the Birch reduction, the classical organic chemical reaction for reductive dehydroxylation of phenolic compounds. The methodology described here represents a novel approach that should help elucidate the mechanisms of other oxidoreductase and redox-activated enzymes.

[Administration of prostaglandin E1 in diabetics and non-diabetic patients with severe Fontaine stage IIb arterial occlusive disease]. / Einsatz von Prostaglandin E1 bei Diabetikern und Nicht-Diabetikern mit schwerer arterieller Verschlusskrankheit im Stadium IIb--IV nach Fontaine.

The success in therapy of prostaglandin E1 intra-arterially and intravenously applied to diabetics and non-diabetics with PAOD, stages IIb-IV according to Fontaine, was scrutinized in a city hospital primarily concerned with geriatrics, metabolism, and angiology. All the patients included were examined in retrospective studies with regard to the improvement of their situations during their stays in hospital and--in addition to that--a part of them prospectively some time after their stays in hospital. In all, 99 patients were included in the retrospective examination of which there were 60 diabetics and 39 non-diabetics. Of the 60 diabetics 53 were in stage IV, 5 in stage III, and 2 in stage IIb while in case of non-diabetics 26 were in stage IV, 10 in stage III, and 3 in stage IIb. 50 of the diabetics and 27 of the non-diabetics were treated intra-arterially. The prospectively orientated post-observation included 38 diabetics and 24 non-diabetics. The results of the retrospective examination revealed a significantly superior efficiency of intra-arterially applied PGE1 compared with the intravenous application, both, with regard to the reduction of needed analgesics and the improvement of the clinical situation. A comparison between intra-arterially treated diabetics and non-diabetics showed a significantly higher rate of success of the diabetics regarding to the improvement of the clinical situation. Concerning the prospective post-examination: due to their PAOD the diabetics took significantly more often analgesics at the time of their post-examination than the non-diabetics. Also concerning the development of their clinical situations the diabetics turned out to have worse results than the non-diabetics, however, the differences were not significant in this case.

Recombinant expression, biochemical characterization, and biological activities of the human MGSA/gro protein.

Melanoma growth stimulatory activity (MGSA) is a mitogenic protein secreted by Hs294T melanoma cells that corresponds to the polypeptide encoded by the human gro gene. The MGSA/gro cDNA has been expressed in mammalian cells and the secreted recombinant factor has been purified. Biochemical and biological characterization shows that the recombinant protein is identical with the natural protein and is devoid of posttranslational glycosylation, sulfation, and phosphorylation. The two C-terminal amino acids are proteolytically removed from the mature recombinant MGSA, indicating a length of 71 instead of the predicted 73 amino acids. The recombinant MGSA is mitogenically active on the Hs294T melanoma cells. The purified MGSA competes with interleukin 8 for binding to neutrophil receptors and exhibits neutrophil chemotactic activity equivalent to that of interleukin 8.

Expression of c-sis and other cellular proto-oncogenes in human sarcoma cell lines and biopsies.

Six biopsies (4 of human fibrosarcomas, 1 of a B-cell lymphoma and 1 of a normal lymph node from a melanoma patient) and 6 cell lines (derived from 5 different human osteosarcomas and from 1 rhabdomyosarcoma), together with control cells, were examined for the expression of c-sis, c-fos and c-myc. The expression of c-sis/PDGF-B-related proteins was also examined in cultured cells (not in biopsies). In situ hybridization studies further showed that the occurrence and level of expression of c-sis mRNA and c-sis/PDGF-B-related proteins were significant in the tumor cells. Expression of c-fos and c-myc mRNA did not correlate with c-sis expression. Southern blot analysis of c-sis, c-fos and c-myc of 20 DNAs of cell lines derived from human sarcoma or biopsies showed an identical pattern for BamH1 and EcoR1 restriction fragments of c-sis (except for 1 fibrosarcoma biopsy), implicating no gene rearrangement as a cause of enhanced proto-oncogene expression. The nucleotide sequence of c-sis is highly homologous to that of the viral v-sis oncogene which is capable of transforming infected cells. We conclude that enhanced expression of c-sis in the sarcomas we have examined is involved in the initiation and/or maintenance of the cell transformed state.

Transmembrane TGF-alpha precursors activate EGF/TGF-alpha receptors.

TGF-alpha and EGF are structurally related factors that bind to and induce tyrosine autophosphorylation of a common receptor. Proteolytic cleavage of the transmembrane TGF-alpha precursor's external domain releases several TGF-alpha species. However, membrane-bound TGF-alpha forms remain on the surface of TGF-alpha-expressing cell lines. To evaluate the biological activity of these forms, we modified two cleavage sites in the TGF-alpha precursor coding sequence, making processing into the 50 amino acid TGF-alpha impossible. Overexpression of this cDNA in a receptor-negative cell line, partial purification, and N-terminal sequence analysis indicate the existence of two transmembrane TGF-alpha forms. These solubilized precursors induce tyrosine autophosphorylation of the EGF/TGF-alpha receptor in intact receptor-overexpressing cells, and anchorage-independent growth of NRK fibroblasts. Cell-cell contact between TGF-alpha precursor-overexpressing cells and cells expressing high numbers of receptors also resulted in receptor activation. These findings suggest a role for transmembrane TGF-alpha forms in intercellular interactions in proliferating tissues.