Modulation of Aβ42 low-n oligomerization using a novel yeast reporter system

Background  

While traditional models of Alzheimer's disease focused on large fibrillar deposits of the Aβ₄₂ amyloid peptide in the brain, recent work suggests that the major pathogenic effects may be attributed to SDS-stable oligomers of Aβ₄₂. These Aβ₄₂ oligomers represent a rational target for therapeutic intervention, yet factors governing their assembly are poorly understood.     

The factor IX phospholipid-binding site is required for calcium-dependent activation of factor IX by factor XIa

To determine the functional role of the metal-dependent conformational changes in Factor IX, two populations of conformation-specific anti-Factor IX antibodies were prepared. Anti-Factor IX X Mg(II) antibodies bind to Factor IX in the presence of Mg(II) and other metal ions, but not in the absence of metal ions. Anti-Factor IX X Ca(II)-specific antibodies bind to Factor IX in the presence of Ca(II) and Sr(II), but not in the presence of Mn(II), Mg(II), and Ba(II). In the presence of a metal ion that induces the conformational transition recognized by the anti-Factor IX X Mg(II) antibodies, the concentrations of CaCl2 and SrCl2 needed for the half-maximal binding of the anti-Factor IX X Ca(II)-specific antibodies to Factor IX were reduced 3- and 20-fold, respectively. Factor IX binding to phospholipid vesicles was inhibited by the Fab fragments of the anti-Factor IX X Ca(II)-specific antibodies, but was not inhibited by the Fab fragments of the anti-Factor IX X Mg(II) antibodies. Factor XIa activation of Factor IX was also inhibited by the Fab fragments of the anti-Factor IX X Ca(II)-specific antibodies, but not by the anti-Factor IX X Mg(II) antibodies. These results support the hypothesis that Factor IX undergoes two metal-dependent conformational transitions: FIX----FIX'----FIX*. The first transition (FIX----FIX') is metal-dependent but cation-nonselective; the second transition (FIX'----FIX*) is metal-selective for Ca(II) or Sr(II). The second transition results in the expression of conformational determinants necessary for membrane binding and the Ca(II)-dependent activation of Factor IX by Factor XIa. These results suggest chemical similarity between a surface of a domain of Factor XIa and phospholipid vesicles, both of which interact with Factor IX in the presence of Ca(II).     

Integration of an aberrant retrotransposon in Saccharomyces cerevisiae.

We describe an atypical composite Ty1 element that apparently resulted from the concurrent integration of two complete elements. A portion of the central region of one of these elements was inverted between two long terminal repeats. Inversions of this type have been detected among unintegrated retroviral circles. It now appears that such intermediates can be incorporated into the genome.     

Analysis of the biomacromolecular architecture of eukaryotic and prokaryotic serine proteases

Summary We have been developing computational approaches to increase our ability to analyze the growing body of three-dimensional structural data with applications centered on the serine proteases and their natural inhibitors and substrates. It is essential that these approaches emphasize the comparison of these macromolecules at the separate levels of secondary, tertiary and quaternary structure. We assume in our analysis that in functionally related macromolecules (i.e., a family of evolutionarily related enzymes), regions of structural and/or physicochemical similarity will exhibit functional similarity; regions that are different in structure and/or physicochemical properties will function differently and, therefore, be the source of observed specificity. It is the intent of our research to encapsulate such knowledge in a form which is capable of observing patterns which may serve as generalizable rules for macrostructural analysis (Liebman, M.N. 1986. Enzyme 36: 150–163), and to serve as the essential tools for the rational design of modified serine proteases and/or their natural inhibitors by the methods available through genetic engineering.     

Structural organization in the serine proteases. I. Macromolecular specificity in limited proteolysis

We have been developing computational approaches to increase our ability to analyze the growing body of three-dimensional structural data with applications centered about the serine proteases. The emphasis of these approaches is to compare and contrast macromolecules at the separate levels of secondary, tertiary, and quaternary structure. Our assumption is that in functionally related molecules, regions of structural and/or physicochemical similarity will exhibit functional similarity; regions that are different in structure and/or physicochemical properties will function differently and, therefore, be the source of specificity. Based on this assumption, the independent observations from studies of these enzymes in solution and in biological systems are combined with the structural observations from X-ray crystallographic analysis. A goal of the present research effort is to probe the biomolecular architecture of the serine proteases to evaluate the role of the three-dimensional structure beyond that of the active site in determining recognition and reactivity determinants for these enzymes, and to determine those principles that might be applied successfully to other enzyme systems as well. Of particular note has been our observation of a macromolecular recognition surface which occurs as a topographical feature outside of the active site and under evolutionary control to produce specificity towards macromolecular substrates and inhibitors. In addition we have established the important role of conformational changes that occur beyond the active site of an enzyme and differentiate between natural and synthetic inhibitor-enzyme interactions. This suggests that the specificity and reactivity determinants of a macromolecule are derived from its architecture and structural organization.     

CGS 10746B: an atypical antipsychotic candidate that selectively decreases dopamine release at behaviorally effective doses

CGS 10746B, a benzothiadiazepine, has a behavioral profile in mice and monkeys similar to the atypical antipsychotic clozapine. Unlike clozapine, CGS 10746B suppresses dopamine neuron firing rates and, when administered at behaviorally effective doses by the oral or intraperitoneal route, decreases neostriatal dopamine release without changing dopamine metabolism or occupying D2 receptors. CGS 10746B is the first atypical antipsychotic candidate that selectively decreases dopamine release.     

The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.      

A translational fidelity mutation in the universally conserved sarcin/ricin domain of 25S yeast ribosomal RNA.

Recent evidence suggests that ribosomal RNAs have functional roles in translation. We describe here a new ribosomal RNA mutation that causes translational suppression and antibiotic resistance in eukaryotic cells. Using random mutagenesis of the cloned ribosomal RNA gene and in vivo selection, we isolated a C --> U mutation in the universally conserved sarcin/ricin domain in Saccharomyces cerevisiae 25S ribosomal RNA. This mutation changes the putative CG pair, which closes the GAGA tetraloop in the sarcin/ricin domain, into a weaker UG pair without eliminating ribosomal sensitivity to ricin. We show that suppression of several UGA, UAG, and frameshift mutations is evident when a portion of the cellular ribosomal RNA contains the C --> U mutation. Cells that contain essentially all mutant ribosomal RNA grow only 10% slower than the wild-type, but show increased suppression as well as resistance to paramomycin, G418, and hygromycin, and sensitivity to cycloheximide. Our results provide genetic evidence for the participation of the sarcin/ricin loop in maintaining translational accuracy and are discussed in terms of a hypothesis that this ribosomal RNA region normally undergoes a conformational change during translation.