Screen for kinases affecting amyloidogenic cleavage by BACE1
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Zusammenfassung
The Amyloid β peptide (Aβ) is suspected to be a causal agent for Alzheimer’s disease (AD). Therefore a screen for kinases downregulating the initial step of its production, the cleavage of the Amyloid Precursor Protein (APP) by Beta-site of APP Cleaving Enzyme 1 (BACE1), was conducted in this study. Briefly, HEK293 cells were colipofected with one of in total 1357 siRNAs against 60% of the human kinome and either an APP construct with only the β-cleavage site left or normally cleavable APP as control. Remaining β-cleavage was for logistic reasons firstly measured with an activity-test for secreted alkaline phosphatase (SEAP) fused to both types of APP and subjected to Aβ-ELISA when interesting. Before the screen, the APP-constructs were characterized in the cell types HEK293 and CGCs with regards to cleavage, especially by BACE1. The screen resulted in 38 hits of which one, Testis Specific Serine Kinase 3, was confirmed once more. In a second, bioinformatic project, an initially suspected APLP-like pseudogenic-like sequence in C3orf52 was refuted. Further, analysis of C3orf52 gene expression data hints on a role in myeloid leukemia. Lastly, the phylogenetic relationship of the APP family paralogs was examined, also in comparison to neighboring gene families, and found in the topology (APLP1)(APLP2/APP).
Zusammenfassung in einer weiteren Sprache
Das Amyloid β-Peptid (Aβ) steht im Verdacht, ursächlich für die Alzheimer-Demenz (AD) zu sein. Daher wurden in dieser Studie Kinasen gesucht, die den ersten Schritt seiner Entstehung, Schnitt des Amyloid Precursor Proteins durch Beta-site of APP Cleaving Enzyme 1 (BACE1), herabregulieren. Dafür wurden HEK293 colipofiziert mit einer von insgesamt 1357 siRNAs gegen 60% des menschlichen Kinoms und entweder einem ausschliesslich β-spaltbarem APP-Konstrukt oder normal spaltbarem APP als Kontrolle. Aus logistischen Gründen wurde die übriggebliebene β-Spaltung zunächst mit einem Aktivitätstest für sekretierbare alkaline Phosphatase (SEAP), das an beide APPs fusioniert war, gemessen und falls interessant noch mit Aβ-ELISA. Vor dem Screen wurden die APP-Konstrukte in den Zelltypen HEK293 und CGC mit Hinblick auf Spaltung, v. a. durch BACE1, charakterisiert. Der Screen resultierte in 38 Hits, von denen einer, Testis Specific Serine Kinase 3, nochmals bestätigt wurde. In einem zweiten, bioinformatischen Projekt wurde eine zunächst vermeintlich APLP-ähnliche pseudogenartige Sequenz in C3orf52 widerlegt. Weiter deutet die Analyse von C3orf52-Genexpressionsdaten auf eine Rolle in myeloider Leukämie hin. Zuletzt wurden die phylogenetischen Beziehungen der APP-Familie-Paraloge untersucht, auch im Vergleich mit benachbarten Genfamilien, und in der Topologie (APLP1)(APLP2/APP) gefunden.
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PENZKOFER, Stephan, 2011. Screen for kinases affecting amyloidogenic cleavage by BACE1 [Dissertation]. Konstanz: University of KonstanzBibTex
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