β-site specific intrabodies to decrease and prevent generation of Alzheimer's Aβ peptide

Abstract

Endoproteolysis of the β-amyloid precursor protein (APP) by β- and γ-secretases generates the toxic amyloid β-peptide (Aβ), which accumulates in the brain of Alzheimer's disease (AD) patients. Here, we established a novel approach to regulate production of Aβ based on intracellular expression of single chain antibodies (intrabodies) raised to an epitope adjacent to the β-secretase cleavage site of human APP. The intrabodies rapidly associated, within the endoplasmic reticulum (ER), with newly synthesized APP. One intrabody remained associated during APP transport along the secretory line, shielded the β-secretase cleavage site and facilitated the alternative, innocuous cleavage operated by α-secretase. Another killer intrabody with an ER retention sequence triggered APP disposal from the ER. The first intrabody drastically inhibited and the second almost abolished generation of Aβ. Intrabodies association with specific substrates rather than with enzymes, may modulate intracellular processes linked to disease with highest specificity and may become instrumental to investigate molecular mechanisms of cellular events.

Inhibiting β-Secretase Activity in Alzheimer's Disease Cell Models with Single-Chain Antibodies Specifically Targeting APP

“…42 An intracellularly expressed scFv derived from a monoclonal antibody raised against the N-terminal Aβ2-6 tetrapeptide EFRH, which also contained an added endoplasmic reticulum retention signal, lowered Aβ levels in cell models. 43 The iBSEC1 scFv also reduces intracellular Aβ levels even though it is added extracellularly. Since iBSEC1 substantially reduces extracellular Aβ levels as well, extracellular addition of the scFv can effectively lower Aβ levels both inside and outside cells.…”

Selective Targeting of Proteins within Secretory Pathway for Endoplasmic Reticulum-associated Degradation

“…Degradins thus represent a novel tool for the induction of specific degradation of target proteins with some interesting features and several possible applications: (i) degradins are very flexible in the design offering the possibility to use as recognition units, scFvs, or other recombinant binders derived from mAbs or selected from different type of libraries, in addition to the use of known ligands or interaction partners, including peptides; (ii) the recognition moiety also can be more complex to include more than one specificity for multiple complex targets, or alternatively different degradins can be co-expressed, each with a different specificity; (iii) depending on the availability of a specific binder, degradins could be useful in targeting protein isoforms with differences in their folding or containing alternative sequences, for instance as a consequence of alternative splicing; (iv) degradins could also be of relevance to degrade disease causing proteins such as the amyloid precursor protein in Alzheimer disease avoiding its appearance on the cell surface (29); the prion protein PrP c , which needs to be transported to the cell surface to be converted to the PrP sc aberrant conformation (30) in different transmissible spongiform encephalopaties like Bovine Spongiform Encephalopathy, Creutzfeld-Jacob disease, and Scrapie (31); or the mutated huntingtin protein in Huntington disease, by reducing the formation of aggregates (32); (v) degradins could be used to target exogenous proteins entering the secretory pathway from pathogens able to persist within the cell in intracellular vacuoles (33,34) such as Slrp from Salmonella typhimurium, major outer membrane protein (MOMP), or IncA from Chlamidia trachomatis, that traffic through the ER and induce ER stress (35,36); (vi) degradins could selectively redirect antigens to proteasome degradation in antigen presenting cells, to increase presentation of antigenderived peptides for activation of antigen-specific cytotoxic CD8 ϩ T-lymphocytes. Other protein knock-out systems that target proteins to the proteasome have been described previously.…”

Targeting antibodies to the cytoplasm

“…The method has been successfully applied to many targets, including human IL2 receptor, ErbB-2 receptor, b-amyloid precursor protein and VCAM1. 6,[17][18][19][20][21] Other subcellular compartments could be targeted by adding suitable signal sequences. 19,22 Functional studies of membrane receptors or secreted proteins ©2 0 1 1 L a n d e s B i o s c i e n c e .…”