Protein mutagenesis with monodispersity-based quality probing: selective inactivation of p53 degradation and DNA-binding properties of HPV E6 oncoprotein

Interpretation of protein mutagenesis experiments requires the ability to distinguish functionally relevant mutations from mutations affecting the structure. When a protein is expressed soluble in bacteria, properly folded mutants are expected to remain soluble whereas misfolded mutants should form insoluble aggregates. However, this rule may fail for proteins fused to highly soluble carrier proteins. In a previous study, we analysed the biophysical status of HPV oncoprotein E6 fused to the C-terminus of maltose-binding protein (MBP) and found that misfolded E6 moieties fused to MBP formed soluble aggregates of high molecular weight. By contrast, preparations of properly folded E6 fused to MBP were monodisperse. Here, we have used this finding to evaluate the quality of 19 MBP-fused E6 site-directed mutants by using a light scattering assay performed in a fluorimeter. This assay guided us to rule out structurally defective mutants and to obtain functionally relevant E6 mutants selectively altered for two molecular activities: degradation of tumour suppressor p53 and DNA recognition.     

Molecular characterization of p53 mutations in primary and secondary liver tumors: diagnostic and therapeutic perspectives

p53 is one of the most mutated genes in human cancer. We have performed the molecular characterization of p53 and have searched for correlations with etiological factors and clinical parameters in primary and secondary liver tumors. A systematic study was carried out, innovative in many respects, to determine the mutational pattern of all 11 exons of p53 and analysis was extended also to exons 1–4 and 9–11 and the exon/intron junctions. Our analyses were performed on case histories of 114 patients from the European area and highlighted p53 mutation patterns different from those reported in the literature for the same tumors. In our case history, different tumors of the same organ showed a different frequency and distribution of mutations. In analyzed tumor types, gene status was a prognostic indicator of survival because patients undergoing liver resection without mutated p53 had a more favorable prognosis than mutated patients. This suggests p53 molecular diagnosis could become a further criterion in the decision for surgery and possible therapies. We describe the ideal conditions for polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and direct sequencing, which we have set in order to optimize yields, sensitivity, and time of what might become a massive molecular screening.     

Two-hybrid-based analysis of protein–protein interactions of the yeast multidrug resistance protein, Pdr5p

The ATP-binding cassette (ABC) transporters are a large family of proteins responsible for the translocation of a variety of compounds across the membranes of both prokaryotes and eukaryotes. The inter-protein and intra-protein interactions in these traffic ATPases are still only poorly understood. In the present study we describe, for the first time, an extensive yeast two-hybrid (Y2H)-based analysis of the interactions of the cytoplasmic loops of the yeast pleiotropic drug resistance (Pdr) protein, Pdr5p, an ABC transporter of Saccharomyces cerevisiae. Four of the major cytosolic loops that have been predicted for this protein [including the two nucleotide-binding domain (NBD)-containing loops and the cytosolic C-terminal region] were subjected to an extensive inter-domain interaction study in addition to being used as baits to identify potential interacting proteins within the cell using the Y2H system. Results of these studies have revealed that the first cytosolic loop (CL1) – containing the first NBD domain – and also the C-terminal region of Pdr5p interact with several candidate proteins. The possibility of an interaction between the CL1 loops of two neighboring Pdr5p molecules was also indicated, which could possibly have implications for dimerization of this protein. Electronic Publication     

The antihypertensive drug carvedilol inhibits the activity of mitochondrial NADH-ubiquinone oxidoreductase

A study is presented on the interaction of carvedilol with mitochondria isolated from several rat organs. It is shown that carvedilol causes a moderate uncoupling effect under non phosphorylating succinate supported respiration of intact mitochondria, as well as a marked inhibition of coupled respiration with NAD-dependent substrates. The inhibitory effect was also found in the bovine heart purified Complex I as well as in experiments with mitochondrial particles, where the individual redox segments of the respiratory chain were analysed. It is also shown that carvedilol, though exhibiting an intrinsic scavenger activity, caused reactive oxygen species to be produced as a consequence of its inhibitory effect on the steady-state respiration. Under these conditions the pro-oxidant activity of carvedilol appears to prevail over its scavenging activity, and a net generation of ROS is promoted.     

A novel family of nuclear transport receptors mediates the export of messenger RNA to the cytoplasm

Fully processed mRNAs are exported to the cytoplasm where they direct protein synthesis. A general feature of mRNA export is that it is an active, receptor-mediated process. The mRNA export receptors are thought to recognize and bind to the mRNA-export cargoes either directly or indirectly (via adaptor proteins) and facilitate their translocation across the central channel of the nuclear pore complex (NPC). On the cytoplasmic side of the NPC, the exported mRNA is released and the receptor returns to the nucleoplasm, without the cargo, to initiate additional rounds of export. Recent, studies in yeast and in higher eukaryotes have led to the elucidation of an evolutionarily conserved pathway for the export of bulk mRNA to the cytoplasm.     

Evaluation of hydrophobicity versus chaperonelike activity of bovine alphaA- and alphaB-crystallin

Calf lens A-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than B-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to A-crystallin than to B-crystallin at room temperature. Bis-ANS binding to both A- and B-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of A-crystallin is lower than that of B-crystallin whereas at higher temperatures, A-crystallin shows significantly higher protection against aggregation of substrate proteins compared to B-crystallin. Therefore, calf lens A-crystallin is more hydrophobic than B-crystallin and chaperone-like activity of -crystallin subunits is not quantitatively related to their hydrophobicity.     

Identification of the site of inhibition of mitogenic signaling by oncogenic ras-p21 by a ras effector peptide

We have previously found that a ras switch 1 domain peptide (PNC-7, residues 35–47) selectively blocks oocyte maturation induced by oncogenic (Val 12–containing) ras-p21 protein and also blocks c-raf–induced oocyte maturation. We now find that oncogenic ras-p21 does not inhibit oocyte maturation of a constitutively activated raf protein (raf BXB) that is lacking most of the first 301 amino terminal amino acids, including the major ras binding domain and accessory ras-binding regions. We also find that a dominant negative raf that completely blocks c-raf–induced maturation likewise does not block raf-BXB–induced maturation. We conclude that PNC-7 blocks ras by binding to the amino-terminal domain of raf and that raf BXB must initiate signal transduction in the cytosol.     

Snake venom hyaluronidase: An evidence for isoforms and extracellular matrix degradation

The present study attempts to establish the isoforms of hyaluronidase enzyme and their possible role in the spreading of toxins during envenomation. Screening of venoms of 15 snakes belonging to three different families revealed varied hyaluronidase activity in ELISA-like assay, but with relatively similar pH and temperature optima. The zymograms of individual venoms showed varied activity banding patterns and indicated the presence of at least two molecular forms of the enzyme. During envenomation, activity of hyaluronidase is considered crucial for the spreading of toxins and is presumed to distort the integrity of extracellular matrix through the degradation of hyaluronic acid in it. This property has been addressed through localization of hyaluronic acid in human skin and muscle tissue sections using the probe, biotinylated hyaluronic acid binding protein. Faint and discontinuous staining pattern of hyaluronidase treated tissue sections over intense staining of untreated tissue sections confirm the selective degradation of hyaluronic acid in extracellular matrix and thus provide an evidence for the spreading property of the enzyme.     

Characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.