Generation and degradation of amyloid β early in the secretory pathway: Monitoring distinct...
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Oral Presentation: Secretuse Process S279 jimJ GENERATION AND DEGRADATION OF AMYLOID p EARLY IN THE SECRETORY PATHWAY: MONITORING DISTINCT PRO- TEASE ACTIVITLES IN A NOVEL IN VITRO y-SECRETASE ASSAY Accumulation of the amyloid p peptide (Ab) into amyloid plaques predominantly in the cerebral cortex and hippocampus is an early and consistent event in the pathogenesis of Alzheimer‘s disease (AD). Since the y-secretase has not been cloned so far, we have developed a cell-free y-secretase assay, in which a purified membrane fraction containing the substrate for the y-secretase, APP-C99, incorporated into the membrane, is utilized to monitor AP40 and AP42 generation and degradation in wfro. The results obtained support the hypothesis that a y-secretase activity, the y-aecretase substrate and the presenilins (PS) are present in a non-acidic subcellular compartment early in the secretory pathway as the Endoplasmatlc Reticulum (ER) and the Golgi apparatus. The inhibitory effect on & now AP generation caused by the known aspartate mutation in PSl (D385N) demonstrate the specificity of the y-secretase cleavage reaction. Data are presented indicating that the y-secretasr is a membrane associated activity with a pH optimum in between 6.8 and 7.4, which can not be inhibited by known protease inhibitors. Furthermore, our results argue for the existence of a @+-dependent proteolytic activity that degrades AP early in the secretory pathway, where it is generated. We consider this novel cell-free y-secretase assay to be an elegant tool for studying the mechanisms underlying the AD pathology in more detail, and, even more important from the pharmaceutical company perspective, for the identification and validation of specific y-secretase inhibitors. This assay allows to distinguish between compounds that might modulate AP secretion (determined by an AP40- and AP42-specific ELBA) through a more general inhibitory mechanism, and those compounds that directly interfere with A@ generation by inhibition of the y-secretase. We are currently focusing on the further optimization of our protocols for purification of membrane fractions enriched m endosomes and lysosomes, respectively. This would enable “9 to monitor and analyze AP generation in distinct subcellular compartments.
Transcript of Generation and degradation of amyloid β early in the secretory pathway: Monitoring distinct...
Oral Presentation: Secretuse Process S279
jimJ GENERATION AND DEGRADATION OF AMYLOID p EARLY IN THE SECRETORY PATHWAY: MONITORING DISTINCT PRO- TEASE ACTIVITLES IN A NOVEL IN VITRO y-SECRETASE ASSAY
Accumulation of the amyloid p peptide (Ab) into amyloid plaques predominantly in the cerebral cortex and hippocampus is an early and consistent event in the pathogenesis of Alzheimer‘s disease (AD). Since the y-secretase has not been cloned so far, we have developed a cell-free y-secretase assay, in which a purified membrane fraction containing the substrate for the y-secretase, APP-C99, incorporated into the membrane, is utilized to monitor AP40 and AP42 generation and degradation in wfro. The results obtained support the hypothesis that a y-secretase activity, the y-aecretase substrate and the presenilins (PS) are present in a non-acidic subcellular compartment early in the secretory pathway as the Endoplasmatlc Reticulum (ER) and the Golgi
apparatus. The inhibitory effect on & now AP generation caused by the known aspartate mutation in PSl (D385N) demonstrate the specificity of the y-secretase cleavage reaction. Data are presented indicating that the y-secretasr is a membrane associated activity with a pH optimum in between 6.8 and 7.4, which can not be inhibited by known protease inhibitors. Furthermore, our results argue for the existence of a @+-dependent proteolytic activity that degrades AP early in the secretory pathway, where it is generated. We consider this novel cell-free y-secretase assay to be an elegant tool for studying the mechanisms underlying the AD pathology in more detail, and, even more important from the pharmaceutical company perspective, for the identification and validation of specific y-secretase inhibitors. This assay allows to distinguish between compounds that might modulate AP secretion (determined by an AP40- and AP42-specific ELBA) through a more general inhibitory mechanism, and those compounds that directly interfere with A@ generation by inhibition of the y-secretase. We are currently focusing on the further optimization of our protocols for purification of membrane fractions enriched m endosomes and lysosomes, respectively. This would enable “9 to monitor and analyze AP generation in distinct subcellular compartments.
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