RTG-ReMPro
Projects
P2 Saftig & Wolf
Regulating the Alzheimer membrane protease BACE1
by targeted protein degradation
Objectives
The major goal of this study is to use the newly developed acute degradation system as an artificial and irreversible protein PTM to identify the direct molecular functions of BACE1 and to validate its suitability as a drug target for Alzheimer's disease. We hypothesise that such a degron system will allow acute and fine-tuned inhibition of BACE1 activity in cellular and in vivo models. We will use it to study the effects of BACE1 degradation on substrate proteins and cellular and organismal phenotypes. Subsequently, we will develop PROTAC molecules to therapeutically downregulate BACE1 protein levels in neuronal cells. These compounds may provide an alternative therapeutic strategy for Alzheimer's Disease (AD) patients.
Aim i) Characterization and expansion of AID system for membrane proteins using BACE1 as example
Aim ii) Cellular and organismal consequences of AID-induced BACE1 degradation
Aim iii) Development of BACE1 specific PROTACs
BACE1 protease topology and proof-of-principle of auxin-induced degradation in HEK cells. (A) Representation of the BACE1-ectodomain linked to the illustrated transmembrane domain and cytoplasmatic tail. (B) Schematic representation of the AID-mediated degradation of BACE1. BACE1 was tagged with the AID tag in cells expressing the plant E3 ligase TIR1. Treatment with auxin induced ubiquitylation of BACE1 by TIR1 and proteasomal degradation. (C) Immunoblot of BACE1. HEK293 cells expressing AID/V5-tagged BACE 1 and TIR1F74G were treated with auxin and the proteasomal inhibitor MG132 for different time points and BACE1 expression was compared to wild-type (naive) cells by immunostaining with antibodies recognizing human BACE1, V5 and GAPDH (loading control). (D) Immunoblot of BACE1. HEK293 cells expressing AID/V5-tagged BACE 1 and TIR1F74G were treated with auxin for different time points and BACE1 expression was compared to wild-type (naive) cells by immunostaining with antibodies recognizing human BACE1, V5 and GAPDH (loading control). (E) ELISA for gp130. The supernatant of HEK293 cells after auxin-induced depletion of AID-tagged BACE1 (Cl32) for 24 h was analysed for levels of cleaved soluble gp130 and compared to wild-type (naive) cells. 5.PhIAA is an indole-3-acetic acid (auxin) derivative.
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