RNA aptamers selectively modulate protein recruitment to the cytoplasmic domain of beta-secretase BACE1 in vitro.
The beta-amyloid peptide (Abeta) is a major component of the Alzheimer's disease (AD)-associated senile plaques and is generated by sequential cleavage of the beta-amyloid precursor protein (APP) by beta-secretase and gamma-secretase. Since BACE1 initiates Abeta generation it represents a valuable target to interfere with Abeta production and treatment of AD. While the enzymatic activity of BACE1 resides in the extracellular domain, the protein also contains a short cytoplasmic tail (B1-CT). This domain serves as a binding site for at least two proteins, the copper chaperone for superoxide dismutase-1 (CCS), and the Golgi-localized, gamma-ear-containing, ADP ribosylation factor-binding (GGA1) protein, and contains a single phosphorylation site. However, the precise role of the B1-CT for the overall biological function of this protein is largely unknown. Functional studies focusing on the activity of this domain would strongly benefit from the availability of domain-specific inhibitors. Here we describe the isolation and characterization of RNA aptamers that selectively target the B1-CT. We show that these RNAs bind to authentic BACE1 and provide evidence that the binding site is restricted to the membrane-proximal half of the C terminus. Aptamer-binding specifically interferes with the recruitment of CCS, but still permits GGA1 association and casein kinase-dependent phosphorylation, consistent with selective binding site targeting within this short peptide. Because phosphorylation and GGA1 binding to B1-CT regulate BACE1 transport, these RNA inhibitors could be applied to investigate B1-CT activity without affecting the subcellular localization of BACE1.
Rentmeister
Andrea
A
LIMES Program Unit Chemical Biology & Medicinal Chemistry, Universität Bonn, 53121 Bonn, Germany.
Bill
Anke
A
Wahle
Tina
T
Walter
Jochen
J
Famulok
Michael
M
D020727:ADP-Ribosylation Factors / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000544:Alzheimer Disease / Q000201:enzymology / Q000235:genetics; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016282:Aspartic Acid Endopeptidases; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry* / Q000201:enzymology*; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D066298:In Vitro Techniques; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D052156:SELEX Aptamer Technique
D033942:Adaptor Proteins, Vesicular Transport; D052157:Aptamers, Nucleotide; C114678:CCS protein, human; C407306:GGA adaptor proteins; D018832:Molecular Chaperones; D011506:Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; D020727:ADP-Ribosylation Factors