9 research outputs found
Changes in readthrough acetylcholinesterase expression modulate amyloid-beta pathology
Alzheimer's disease has long been known to involve cholinergic deficits, but the linkage between cholinergic gene expression and the Alzheimer's disease amyloid pathology has remained incompletely understood. One known link involves synaptic acetylcholinesterase (AChE-S), shown to accelerate amyloid fibrils formation. Here, we report that the ‘Readthrough' AChE-R splice variant, which differs from AChE-S in its 26 C-terminal residues, inversely exerts neuroprotective effects from amyloid β (Aβ) induced toxicity. In vitro, highly purified AChE-R dose-dependently suppressed the formation of insoluble Aβ oligomers and fibrils and abolished Aβ toxicity to cultured cells, competing with the prevalent AChE-S protein which facilitates these processes. In vivo, double transgenic APPsw/AChE-R mice showed lower plaque burden, fewer reactive astrocytes and less dendritic damage than single APPsw mice, inverse to reported acceleration of these features in double APPsw/AChE-S mice. In hippocampi from Alzheimer's disease patients (n = 10), dentate gyrus neurons showed significantly elevated AChE-R mRNA and reduced AChE-S mRNA. However, immunoblot analyses revealed drastic reductions in the levels of intact AChE-R protein, suggesting that its selective loss in the Alzheimer's disease brain exacerbates the Aβ-induced damages and revealing a previously unforeseen linkage between cholinergic and amyloidogenic event
N-Acetylcholinesterase-Induced Apoptosis in Alzheimer's Disease
Background: Alzheimer’s disease (AD) involves loss of cholinergic neurons and Tau protein hyper-phosphorylation. Here, we report that overexpression of an N-terminally extended ‘‘synaptic’ ’ acetylcholinesterase variant, N-AChE-S is causally involved in both these phenomena. Methodology and Principal Findings: In transfected primary brain cultures, N-AChE-S induced cell death, morphological impairments and caspase 3 activation. Rapid internalization of fluorescently labeled fasciculin-2 to N-AChE-S transfected cells indicated membranal localization. In cultured cell lines, N-AChE-S transfection activated the Tau kinase GSK3, induced Tau hyper-phosphorylation and caused apoptosis. N-AChE-S-induced cell death was suppressible by inhibiting GSK3 or caspases, by enforced overexpression of the anti-apoptotic Bcl2 proteins, or by AChE inhibition or silencing. Moreover, inherent N-AChE-S was upregulated by stressors inducing protein misfolding and calcium imbalances, both characteristic of AD; and in cortical tissues from AD patients, N-AChE-S overexpression coincides with Tau hyper-phosphorylation. Conclusions: Together, these findings attribute an apoptogenic role to N-AChE-S and outline a potential value to ACh
Temperature-Controlled Activity of DnaK−DnaJ−GrpE Chaperones: Protein-Folding Arrest and Recovery during and after Heat Shock Depends on the Substrate Protein and the GrpE Concentration †
Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol–overexpressed molecular chaperones
When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species. In this study, metal affinity purification (immobilized metal ion affinity chromatography [IMAC]), confirmed by resistance to trypsin digestion, was used to distinguish soluble aggregates from soluble nativelike proteins. Salt-induced accumulation of osmolytes during induced protein synthesis significantly improved IMAC yields of folding-recalcitrant proteins. Yet, the highest yields were obtained with cells coexpressing plasmid-encoded molecular chaperones DnaK-DnaJ-GrpE, ClpB, GroEL-GroES, and IbpA/B. Addition of the membrane fluidizer heat shock–inducer benzyl alcohol (BA) to the bacterial medium resulted in similar high yields as with plasmid-mediated chaperone coexpression. Our results suggest that simple BA-mediated induction of endogenous chaperones can substitute for the more demanding approach of chaperone coexpression. Combined strategies of osmolyte-induced native folding with heat-, BA-, or plasmid-induced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes
Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses.
Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes
Highly-dispersed ultrafine Pt nanoparticles on microemulsion-mediated TiO2 for production of hydrogen and valuable chemicals via oxidative photo-dehydrogenation of glycerol
The oxidative photo-dehydrogenation of glycerol to produce H2 and other valuable chemicals was studied using different materials. In particular, Pt nanoparticles were deposited on microemulsion-synthesized TiO2 via surface organometallic chemistry (SOMC) and compared with photocatalysts obtained using more conventional methods. Well-defined Pt(II) single-site titania-grafted were prepared reacting the surface hydroxyl groups of TiO2 nano-oxides with the organometallic Pt(COD)Me2 complex. Sample reduction under H2 generated ultrafine Pt nanoparticles well-dispersed on titania surface. Its performance under simulated solar light showed superior activity when compared to analogous Pt-containing catalysts prepared by other methods. Improved dispersion of Pt metal on titania surface was among the primary reasons of a better overall activity, providing relatively high rates of hydrogen productivity. Moreover, an increase of glyceraldehyde productivity in liquid phase was observed with the increase of Pt dispersion, demonstrating that the metal dispersion can strongly affect the selectivity of chemicals produced in the reaction. Comparison with state of the art shows that the present material exhibits excellent performance for a combined positive effect of the high specific surface area of titania prepared by microemulsion, giving access to the increased densities of active sites and the high dispersion of Pt nanoparticles given by the SOMC technique