Wild‐type Shadoo proteins convert to amyloid‐like forms under native conditions

Abstract

J. Neurochem. (2010) 10.1111/j.1471‐4159.2010.06575.x Abstract The cellular prion protein PrPC refolds into a β‐sheet enriched, infectivity‐associated form called PrPSc. Shadoo (Sho) is a newly discovered glycoprotein that is also expressed in the adult brain. Wild type (wt) mouse Sho consists of an arginine‐rich region, a hydrophobic central domain of five tandem A/LAAG amino acid repeats R1–R5 with similarity to the hydrophobic domain of PrPC, and a C‐terminal domain with one N‐linked carbohydrate. As some alanine‐rich proteins and PrP with a shortened C‐terminal domain form amyloid we investigated conformational properties of wt Sho and polymorphic variants with insertion/deletions centered on R3. Recombinant mouse and sheep Sho converted to an amyloid‐like form without recourse to chemical denaturation or acidification. For wt proteins this transition was marked by increased thioflavin T binding, Congo red staining, presence of fibrillar structures by electron microscopy, formation of sodium dodecyl sulfate‐resistant complexes and the generation of a C‐terminal proteinase K resistant core of 5–8 kDa. Variant Sho proteins differing within the R1–R5 region exhibited most but not all of these properties. Our studies define a proteinase K ‐resistant signature fragment for the amyloid fold of Sho and raise the question of a physiological role for this form of the wt protein.

Amyloid Assemblies of Influenza A Virus PB1-F2 Protein Damage Membrane and Induce Cytotoxicity

“…This spontaneous conformational switch additionally points out that the C-terminal part of PB1-F2 is initially deprived of any secondary structure at these pH values. Indeed, whereas most ␣-helical proteins such as ␤ 2 -microglobulin, lysozyme, or prion protein need some chemical or thermal treatment to convert into amyloid structures in vitro (39 -41), natively disordered proteins as A␤, Shadoo, ␣-synuclain may switch to amyloid-like forms without recourse to denaturation treatment (42)(43)(44)(45).…”

Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrP C -deficiency

“…In terms of known PrP-like molecules, Doppel is barely expressed in the CNS in wild-type mice (35,36) and can be excluded from consideration. However, because Sho is expressed in the CNS (21 and present study) and has a number of PrP C -like biochemical properties (21,25,37), it is considered as a candidate for π. Indeed, the report of embryonic lethality from knockdown of Sho in Prnp 0/0 embryos (26) added impetus to this notion.…”

The α-Helical Structure of Prodomains Promotes Translocation of Intrinsically Disordered Neuropeptide Hormones into the Endoplasmic Reticulum

“…As examples for ␣-helical domains, we used (i) the prodomain of Som (aa 25-88), (ii) the C-terminal domain of the prion protein (aa 173-219 or aa 130 -230) (52)(53)(54), and (iii) a synthetically designed ␣-helix (25). To test the effect of intrinsically disordered domains, we used part of the N-terminal domain of the prion protein (aa 23-115) (52) or shadoo (aa 25-125) (55,56). N2a cells were transiently transfected with the different constructs, and conditioned medium was analyzed by Western blotting.…”