Folding of Tetrameric p53: Oligomerization and Tumorigenic Mutations Induce Misfolding and Loss of Function

The physiologically active form of p53 consists of a tetramer of four identical 393-amino-acid subunits associated via their tetramerization domains (TDs; residues 325-355). One in two human tumors contains a point mutation in the DNA binding domain (DBD) of p53 (residues 94-312). Most existing studies on the effects of these mutations on p53 structure and function have been carried out on the isolated DBD fragment, which is monomeric. Recent structural evidence, however, suggests that DBDs may interact with each other in full-length tetrameric forms of p53. Here, we investigate the effects of tumorigenic DBD mutations on the folding of p53 in its tetrameric form. We employ the construct consisting of DBD and TD (amino acids 94-360). We characterize the stability and conformational state of the tumorigenic DBD mutants R248Q, R249S, and R282Q using equilibrium denaturation and functional assays. Destabilizing mutations cause DBD to misfold when it is part of the p53 tetramer, but not when it is monomeric. This conformation is populated under moderately destabilizing conditions (10 °C in 2 M urea, and at physiological temperature in the absence of denaturant). Under those same conditions, it is not present in the isolated DBD fragment or in the presence of the TD mutation L344P, which abolishes tetramerization. Misfolding appears to involve intramolecular DBD-DBD association within a single tetrameric molecule. This association is promoted by destabilization of DBD (caused by mutation or elevated temperature) and by the high local DBD concentration enforced by tetramerization of TD. Disrupting the nonnative DBD-DBD interaction or transiently inhibiting tetramerization and allowing p53 to fold as a monomer may be potential strategies for pharmacological intervention in cancer.     

Studies on the forms of iron-transferrin released from rabbit reticulocytes

Measurement of the distribution of the four species of transferrin, viz, apotransferrin, diferric transferrin and the two monoferric transferrin, before and after incubation of iron-rich rabbit transferrin with rabbit reticulocytes showed that not all transferrin released from the cells were in the form of apotransferrin. Instead, a mixture of all four species of the protein was released with apotransferrin and C-terminal monoferric transferrin being the major fractions. The buffer solution containing 125I-labelled transferrin showed a continuous gain in percentages in apotransferrin and C-terminal monoferric transferrin after each incubation with reticulocytes. The N-terminal monoferric transferrin, however, remained unchanged suggesting that in the process of transferrin uptake by cells, the diferric transferrin releases its iron from the acid-labile site at N-domain first before the other iron from the acid-stable site.     

Effects of hypophysectomy and ACTH on opiate tolerance and physical dependence.

In opiate-naive hypophysectomized rats, the enhanced potency of parenterally injected morphine was almost completely normalized by chronic ACTH in doses that maintained adrenal weight. Following morphine pellet implantation, the amount of morphine required for antinociception, catalepsy, and colonic temperature effects in tolerant rats was identical among hypophysectomized and sham-operated rats whether or not chronic ACTH was administered. However, brain levels of morphine were greater in tolerant, hypophysectomized rats, an effect blocked by chronic ACTH. Hypophysectomy thus appears to enhance the magnitude of tolerance development by a mechanism that is reversed by doses of ACTH that maintain adrenal weight. On the other hand, in sham-control rats this dose and schedule of ACTH were without effect on morphine potency in opiate-naive or tolerant rats or on brain levels of morphine. In general, hypophysectomy and/or ACTH did not modify the intensity of abstinence signs following naloxone-precipitated withdrawal. Therefore, little evidence was obtained to suggest a direct, commanding role of the pituitary in chronic opiate effects. Instead, a secondary adrenal involvement may be important.     

Cytolysin A-mediated protein exportation efficiency and its role in enhancing the fitness of live recombinant Salmonella Typhi vaccine strain

The genetic fusion of cytolysin A (clyA) to heterologous antigen expressed in live Salmonella vector demonstrated efficient translocation into periplasmic space and extracellular medium. Accumulating evidence has shown that clyA-mediated antigen delivery improved growth fitness and enhanced immunogenicity of live vector vaccine, but the factors influencing this protein exportation has not been investigated. In this study, Toxoplasma gondii antigen fused at C-terminal of clyA protein was expressed in live S. Typhi vector via both plasmid and chromosomal-based expressions. The bivalent strains showed comparable growth rates as monovalent strains, but in varies antigen exportation efficiency. ClyA-fusion antigen with positive charges was translocated to the extracellular spaces, whereas those with negative charges were retained in the cytoplasm. Furthermore, excessive cellular resources expenditure on antigen expression, especially antigen with larger size, could limit the clyA-fusion antigen exportation, resulting in undesirable metabolic burden that eventually affects the growth fitness. Altogether, the present work indicates potential linkage of factors mainly on antigen properties and expression platforms that may affect clyA-mediated antigen delivery to enhance the growth fitness of live vector strain.     

Immunoreactive peptides related to dynorphin B (=rimorphin) in the rat brain

We have developed a radioimmunoassay for synthetic dynorphin B, a novel opioid tridecapeptide, which shares a common precursor molecule with dynorphin_1–17 (=dynorphin A) and the neo-endorphins. The levels of immunoreactivity towards this peptide in rat brain and pituitary show a pattern quantitatively and qualitatively similar to those found for dynorphin A and -neo-endorphin in earlier studies. The antiserum used was highly specific with only dynorphin-32 and dynorphin B-29, both of which contain the dynorphin B sequence, showing substantial cross-reactivity. Gel filtration of whole rat brain extracts in combination with HPLC analysis provide strong evidence for the existence of these latter two peptides in rat brain.     

Determination of chlorpromazine and its major metabolites by gas chromatography/mass spectrometry: application to biological fluids

A method for the quantitative determination of chlorpromazine and five of its major metabolites in a single sample of biological fluid in the ng/ml range has been developed utilizing gas chromatography/mass spectrometry with selected ion recording. The assay is highly specific and quantification is accomplished by an inverse stable isotope dilution technique, using deuterium-labeled variants of the compounds as internal standards. In this way the concentrations of chlorpromazine and five of its major metabolites (the sulfoxide, the N-oxide, the monodemethylated, the didemethylated, and the 7-hydroxylated compounds) can be determined in biological fluids. Levels in humans have been measured both in plasma and in red blood cells and are compared to those found in related in vitro studies.     

A model for opiate-receptor interactions: mechanism of opiate-cerebroside sulfate interaction.

Narcotic analgetics were shown to bind cerebroside sulfate (CS) with high affinity. The binding correlated well with their pharmacological potency. In order to understand opiate receptor interaction at the molecular level, we have proposed the use of CS as a model opiate receptor. In these studies, our data indicate that the binding of opiates is determined by the heptane solubility of the drugs and their affinity to CS. The affinity of the agonist to CS is higher than that of its corresponding antagonist. The difference in affinity between an agonist and its corresponding antagonist is mainly due to the strength of electrostatic bond formed between the protonated nitrogen of the drug and the sulfate group of CS. Furthermore, we have concluded that narcotic agonist-CS complexes are more hydrophobic (intimate ion pairs formation) while the antagonist-CS complexes are more hydrophilic (hydrated ion pairs) in nature.