Assessing amyloidogenic potential of urinary proteins from women with preeclampsia using yeast-based assay

BACKGROUND: Preeclampsia is a serious pregnancy disorder that arises after 20 weeks’ gestation and is characterized by hypertension, proteinuria and edema; it can progress to eclampsia with severe complications and death. Several studies have reported amyloid like aggregates in placenta and urine from preeclampsia patients that stain with Congo red. Mass spectrometry of these aggregates identified Albumin, Ceruloplasmin, Interferon alpha inducible protein 6, Serotransferrin, Alpha 1 antitrypsin, immunoglobulin light chains (κ) and Aβ peptides. Except for Aβ, and immunoglobulin light chains, the ability of these proteins to form amyloids in vivo has not been demonstrated.

AIM: To evaluate the in vivo amyloidogenic potential of proteins identified in preeclampsia associated urinary aggregates using the yeast based assay.

METHODS: Candidate human proteins (including Aβ42 as positive control) were cloned in frame with the N-terminal prion domain of S. cerevisiae Sup35 (Sup35N) into a yeast expression plasmid. Constructs were under control the copper inducible CUP1 promoter. The constructs were transformed into a S. cerevisiae ade1-14 strain (nonsense mutation in ADE1) and lacking endogenous prions. Expression was induced by growth colonies on −Ura medium containing 150 µM CuSO₄ for 48 h. Colonies were then replica plated to adenine deficient (−Ade) medium, and growth was monitored up to 21 days as a readout of Sup35 conversion to the [PSI⁺] prion and of the amyloidogenicity of the Sup35N fusion.

RESULTS: Analysis indicates that none of the human proteins tested—except the Aβ42 peptide—have demonstrated amyloidogenic potential in vivo.

CONCLUSION: None of the full-length human proteins detected in urine from preeclampsia patients–albumin, ceruloplasmin, IFN-α-inducible protein 6, serotransferrin, and alpha-1-antitrypsin showed amyloidogenic activity in a yeast-based phenotypic assay; only Aβ42 functioned as an effective seed. Future work will assess amyloidogenicity of individual domains of these proteins.