Identification and isolation of a hyperphosphorylated, conformationally changed intermediate of human protein tau expressed in yeast.
Hyperphosphorylation and aggregation of protein tau are typical for neurodegenerative tauopathies, including Alzheimer's disease (AD). We demonstrate here that human tau expressed in yeast acquired pathological phosphoepitopes, assumed a pathological conformation, and formed aggregates. These processes were modulated by yeast kinases Mds1 and Pho85, orthologues of GSK-3beta and cdk5, respectively. Surprisingly, inactivation of Pho85 increased phosphorylation of tau-4R, concomitant with increased conformational change defined by antibody MC1 and a 40-fold increase in aggregation. Soluble protein tau, purified from yeast lacking PHO85, spontaneously and rapidly formed tau filaments in vitro. Further fractionation of tau by anion-exchange chromatography yielded a hyperphosphorylated monomeric subfraction, termed hP-tau/MC1, with slow electrophoretic mobility and enriched with all major epitopes, including MC1. Isolated hP-tau/MC1 vastly accelerated in vitro aggregation of wild-type tau-4R, demonstrating its functional capacity to initiate aggregation, as well as its structural stability. Combined, this novel yeast model recapitulates hyperphosphorylation, conformation, and aggregation of protein tau, provides insight in molecular changes crucial in tauopathies, offers a source for isolation of modified protein tau, and has potential for identification of modulating compounds and genes.
Vandebroek
Tom
T
Experimental Genetics Group, Leuven, Belgium.
Vanhelmont
Thomas
T
Terwel
Dick
D
Borghgraef
Peter
P
Lemaire
Katleen
K
Snauwaert
Johan
J
Wera
Stefaan
S
Van Leuven
Fred
F
Winderickx
Joris
J
D000906:Antibodies; D006801:Humans; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D016875:tau Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D016875:tau Proteins; D011494:Protein Kinases