Structural elements regulating amyloidogenesis: a cholinesterase model system

Polymerization into amyloid fibrils is a crucial step in the pathogenesis of neurodegenerative syndromes. Amyloid assembly is governed by properties of the sequence backbone and specific side-chain interactions, since fibrils from unrelated sequences possess similar structures and morphologies. Therefore, characterization of the structural determinants driving amyloid aggregation is of fundamental importance. We investigated the forces involved in the amyloid assembly of a model peptide derived from the oligomerization domain of acetylcholinesterase (AChE), AChE(586-599), through the effect of single point mutations on beta-sheet propensity, conformation, fibrilization, surfactant activity, oligomerization and fibril morphology. AChE(586-599) was chosen due to its fibrilization tractability and AChE involvement in Alzheimer's disease. The results revealed how specific regions and residues can control AChE(586-599) assembly. Hydrophobic and/or aromatic residues were crucial for maintaining a high beta-strand propensity, for the conformational transition to beta-sheet, and for the first stage of aggregation. We also demonstrated that positively charged side-chains might be involved in electrostatic interactions, which could control the transition to beta-sheet, the oligomerization and assembly stability. Further interactions were also found to participate in the assembly. We showed that some residues were important for AChE(586-599) surfactant activity and that amyloid assembly might preferentially occur at an air-water interface. Consistently with the experimental observations and assembly models for other amyloid systems, we propose a model for AChE(586-599) assembly in which a steric-zipper formed through specific interactions (hydrophobic, electrostatic, cation-pi, SH-aromatic, metal chelation and polar-polar) would maintain the beta-sheets together. We also propose that the stacking between the strands in the beta-sheets along the fiber axis could be stabilized through pi-pi interactions and metal chelation. The dissection of the specific molecular recognition driving AChE(586-599) amyloid assembly has provided further knowledge on such poorly understood and complicated process, which could be applied to protein folding and the targeting of amyloid diseases.     

Differential gene expression and epiregulation of alpha zein gene copies in maize haplotypes

Multigenic traits are very common in plants and cause diversity. Nutritional quality is such a trait, and one of its factors is the composition and relative expression of storage protein genes. In maize, they represent a medium-size gene family distributed over several chromosomes and unlinked locations. Two inbreds, B73 and BSSS53, both from the Iowa Stiff Stock Synthetic collection, have been selected to analyze allelic and non-allelic variability in these regions that span between 80-500 kb of chromosomal DNA. Genes were copied to unlinked sites before and after allotetraploidization of maize, but before transposition enlarged intergenic regions in a haplotype-specific manner. Once genes are copied, expression of donor genes is reduced relative to new copies. Epigenetic regulation seems to contribute to silencing older copies, because some of them can be reactivated when endosperm is maintained as cultured cells, indicating that copy number variation might contribute to a reserve of gene copies. Bisulfite sequencing of the promoter region also shows different methylation patterns among gene clusters as well as differences between tissues, suggesting a possible position effect on regulatory mechanisms as a result of inserting copies at unlinked locations. The observations offer a potential paradigm for how different gene families evolve and the impact this has on their expression and regulation of their members.     

Integrated computer system for regulating eukaryotic gene expression

The review describes several modules of the GeneExpress integrated computer system concerning the regulation of gene expression in eukaryotes. Approaches to the presentation of experimental data in databases are considered. The employment of GeneExpress in computer analysis and modeling of the organization and function of genetic systems is illustrated with examples. GeneExpress is available at http://wwwmgs.bionet.nsc.ru/mgs/gnw/.     

Control elements of muscarinic receptor gene expression

Studies describing the structures of the M1, M2 and M4 muscarinic acetylcholine receptors (mAChR) genes and the genetic elements that control their expression are reviewed. In particular, we focus on the role of the neuron-restrictive silencer element/restriction element-1 (NRSE/RE-1) in the regulation of the M4 mAChR gene. The NRSE/RE-1 was first identified as a genetic control element that prevents the expression of the SCG-10 and type II sodium channel (NaII) genes in non-neuronal cells in culture. The NRSE/RE-1 inhibits gene expression by binding the repressor/silencer protein NRSF/REST, which is present in many non-neuronal cell lines and tissues. Our studies show that although the expression of the M4 mAChR gene is inhibited by NRSF/REST, this inhibition is not always complete. Rather, the efficiency of silencing by NRSF/REST is different in different cells. A plausible explanation for this differential silencing is that the NRSF/RE-1 interacts with distinct sets of promoter binding proteins in different types of cells. We hypothesize that modulation of NRSF/REST silencing activity by these proteins contributes to the cell-specific pattern of expression of the M4 mAChR in neuronal and non-neuronal cells. Recent studies that suggest a more complex role for the NRSE/RE-1 in regulating gene expression are also discussed.     

DNA sequences regulating human beta globin gene expression.

Human delta globin is expressed at approximately 1-2% of the level of human beta globin in erythroid cells despite the marked homology between these two globins. To determine the DNA sequences responsible for this effect, delta and beta globin genes and fusion products of these genes constructed in vitro were transfected and expressed in HeLa cells. The results indicate that when the small intervening sequence of the beta gene (beta IVS 1) is replaced by delta IVS 1, expression of the chimeric gene is the same as that of the normal beta globin gene. By contrast, when the large intervening sequence of the beta gene (beta IVS 2) is replaced by delta IVS 2, expression of the chimeric gene is markedly reduced. These results suggest that there are signals within IVS 2 of the delta and beta genes which affect their relative expression.     

Molecular-genetic mechanisms regulating tissue-specific expression of the drosophilaestS gene

A study was made of theDrosophila melanogaster est6 andD. virilis estS genes for tissue-specific esterase, and their expression at various stages of development was characterized. The former has one promoter and is expressed in the seminal ducts, whereas the latter has two promoters and is expressed in the seminal bulbs. In transgenicD. melanogaster, estS was expressed in the seminal bulbs, as observed in the donor. A region adjacent to the structural gene proved responsible for its expression in the seminal bulbs. TransgenicD. melanogaster lines were also obtained with constructs containing various fragments of theestS regulatory region and thelacZ reporter gene. Histochemical analysis with X-Gal staining allowed identification of a region that inhibitsestS expression in all organs other than seminal bulbs. An esterase S homolog was found in a marine mollusk.     

Membrane sites regulating developmental gene expression in Dictyostelium discoideum

Postaggregative gene expression in Dictyostelium discoideum requires cell contact. Polyspecific monovalent antibodies (Fab) prepared from sera raised against membranes of aggregation- and postaggregation-stage cells were used to probe the cell interactions that induce rapid postaggregative synthesis of UDP-glucose pyrophosphorylase. When cells of strain V12M2 were dissociated after 8 hr of development and replated in the presence of immune Fab, both reaggregation and pyrophosphorylase synthesis were blocked. Fab neutralized by incubation with EDTA-high salt extracts of cells developed for 3 hr blocked pyrophosphorylase synthesis but not reaggregation. Therefore, some cell-surface components that regulate pyrophosphorylase synthesis (called E sites) are antigenically distinct from those required for reaggregation. The Fab provides a means to assay E sites during their purification. Addition of 10(-3) M cyclic AMP or cyclic GMP enabled the cells to bypass the blocking of E sites by Fab; pyrophosphorylase was synthesized in the absence of reaggregation. We hypothesize that E sites function by raising the level of intracellular cyclic AMP.