The overproduction of β-amyloid (Aβ) fragments in transgenic APPswe/PS1dE9 mice results in formation of amyloid deposits in the cerebral cortex and hippocampus starting around four months of age and leading to cognitive impairment much later. We have previously found an age and transgene-dependent weakening of muscarinic receptor-mediated transmission that was not present in young (6-10-week-old) animals but preceded both amyloid deposits and cognitive deficits. Now we investigated immediate and prolonged in vitro effects of non-aggregated Aβ(1-42) on coupling of individual muscarinic receptor subtypes expressed in CHO (Chinese hamster ovary) cells and their underlying mechanisms. Immediate application of 1 μM Aβ(1-42) had no effect on the binding of the muscarinic antagonist N-methylscopolamine or the agonist carbachol. In contrast, 4-day treatment of CHO cells expressing the M1 muscarinic receptor with 100 nM Aβ(1-42) significantly changed the binding characteristics of the muscarinic agonist carbachol and reduced the extent of the M1 receptor-stimulated breakdown of phosphatidylinositol while it did not demonstrate overt toxic effects. The treatment had no influence on the expression of either G-proteins or muscarinic receptors. In concert, we found no change in the gene expression of muscarinic receptor subtypes and gene or protein expression of the G(s), G(q/11), and G(i/o) G-proteins in the cerebral cortex of young adult APPswe/PS1dE9 mice that demonstrate high concentrations of soluble Aβ(1-42) and impaired muscarinic receptor-mediated G-protein activation. Our results provide strong evidence that the initial injurious effects of Aβ(1-42) on M1 muscarinic receptor-mediated transmissionis is due to compromised coupling of the receptor with G(q/11) G-protein.

• MeSH
• amyloidní beta-protein antagonisté a inhibitory   metabolismus   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• křečci praví MeSH
• kultivované buňky MeSH
• lidé MeSH
• myši transgenní MeSH
• myši MeSH
• peptidové fragmenty antagonisté a inhibitory   metabolismus   MeSH
• receptor muskarinový M1 metabolismus   MeSH
• receptory spřažené s G-proteiny antagonisté a inhibitory   metabolismus   MeSH
• rozpřahující látky farmakologie   MeSH
• vazba proteinů účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Propeptide blocks the active site in the inactive zymogen of cathepsin D and is cleaved off during zymogen activation. We have designed a set of peptidic fragments derived from the propeptide structure and evaluated their inhibitory potency against mature cathepsin D using a kinetic assay. Our mapping of the cathepsin D propeptide indicated two domains in the propeptide involved in the inhibitory interaction with the enzyme core: the active site "anchor" domain and the N-terminus of the propeptide. The latter plays a dominant role in propeptide inhibition (nanomolar Ki), and its high-affinity binding was corroborated by fluorescence polarization measurements. In addition to the inhibitory domains of propeptide, a fragment derived from the N-terminus of mature cathepsin D displayed inhibition. This finding supports its proposed regulatory function. The interaction mechanisms of the identified inhibitory domains were characterized by determining their modes of inhibition as well as by spatial modeling of the propeptide in the zymogen molecule. The inhibitory interaction of the N-terminal propeptide domain was abolished in the presence of sulfated polysaccharides, which interact with basic propeptide residues. The inhibitory potency of the active site anchor domain was affected by the Ala38pVal substitution, a propeptide polymorphism reported to be associated with the pathology of Alzheimer's disease. We infer that propeptide is a sensitive tethered ligand that allows for complex modulation of cathepsin D zymogen activation.

• MeSH
• aminokyselinové motivy MeSH
• financování organizované MeSH
• glykosaminoglykany metabolismus   MeSH
• inhibitory proteas chemická syntéza   metabolismus   MeSH
• katalytická doména MeSH
• kathepsin D antagonisté a inhibitory   chemie   metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• peptidové fragmenty antagonisté a inhibitory   chemická syntéza   metabolismus   MeSH
• peptidové mapování MeSH
• prekurzory enzymů antagonisté a inhibitory   chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH