Abstract
The structurally disordered N-terminal half of the prion protein (PrPC) is constitutively released into the extracellular space by an endogenous proteolytic cleavage event. Once liberated, this N1 fragment acts neuroprotective in ischemic conditions and interferes with toxic peptides associated with neurodegenerative diseases, such as amyloid-beta (Aβ) in Alzheimer’s disease. Since analog protective effects of N1 in prion diseases, such as Creutzfeldt-Jakob disease, have not been studied, and given that the protease releasing N1 has not been identified to date, we have generated and characterized transgenic mice overexpressing N1 (TgN1). Upon intracerebral inoculation of TgN1 mice with prions, no protective effects were observed at the levels of survival, clinical course, neuropathological, or molecular assessment. Likewise, primary neurons of these mice did not show protection against Aβ toxicity. Our biochemical and morphological analyses revealed that this lack of protective effects is seemingly due to an impaired ER translocation of the disordered N1 resulting in its cytosolic retention with an uncleaved signal peptide. Thus, TgN1 mice represent the first animal model to prove the inefficient ER translocation of intrinsically disordered domains (IDD). In contrast to earlier studies, our data challenge roles of cytoplasmic N1 as a cell penetrating peptide or as a potent “anti-prion” agent. Lastly, our study highlights both the importance of structured domains in the nascent chain for proteins to be translocated and aspects to be considered when devising novel N1-based therapeutic approaches against neurodegenerative diseases.
Highlights
The prion protein (PrPC), a glycoprotein with high expression levels and biological relevance in the nervous system, isMol Neurobiol (2020) 57:2812–2829 composed of two structurally very dissimilar parts [1,2,3]
Overexpression of the transgene was confirmed on the genetic level by copy number analysis (ΔΔCt = − 3.913, corresponding to a fold change of 15; data not shown) and on the mRNA level for cerebellum (TgN1: 3.41 ± 0.58; WT set to 1.00 ± 0.15; n = 3; SEM; Fig. 2c) and forebrain (TgN1: 2.37 ± 0.48; WT set to 1.00 ± 0.24; n = 3; SEM; Fig. 2d)
Western blot (WB) analysis of freshly prepared forebrain homogenates clearly proved overexpression of N1 that was even more pronounced in aged mice, whereas levels of full-length PrPC and an N-terminal PrP
Summary
The prion protein (PrPC), a glycoprotein with high expression levels and biological relevance in the nervous system, isMol Neurobiol (2020) 57:2812–2829 composed of two structurally very dissimilar parts [1,2,3]. The N-terminal half of the molecule is highly flexible and lacks structural features [5], representing a prototype of so-called intrinsically disordered domains/proteins (IDD/ IDP, Fig. 1) [6] Such IDDs inherently miss folded parts as preformed “binding interfaces”, they are able to initiate transient high-affinity interactions with other proteins [7]. As part of the membrane-bound full-length PrPC, the N-terminal half is considered as a flexible molecular sensor of the extracellular milieu [8] that may transiently adopt a particular shape upon binding of specific ligands [9] Several of those interacting ligands have been described, among them divalent cations as well as various proteins/ peptides of physiological or pathological relevance [8, 10,11,12,13]. The N-terminal part is critical for the initial
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