A universal method for detection of amyloidogenic misfolded proteins.

Diseases associated with the misfolding of endogenous proteins, such as Alzheimer's disease and type II diabetes, are becoming increasingly prevalent. The pathophysiology of these diseases is not totally understood, but mounting evidence suggests that the misfolded protein aggregates themselves may be toxic to cells and serve as key mediators of cell death. As such, an assay that can detect aggregates in a sensitive and selective fashion could provide the basis for early detection of disease, before cellular damage occurs. Here we report the evolution of a reagent that can selectively capture diverse misfolded proteins by interacting with a common supramolecular feature of protein aggregates. By coupling this enrichment tool with protein specific immunoassays, diverse misfolded proteins and sub-femtomole amounts of oligomeric aggregates can be detected in complex biological matrices. We anticipate that this near-universal approach for quantitative misfolded protein detection will become a useful research tool for better understanding amyloidogenic protein pathology as well as serve as the basis for early detection of misfolded protein diseases.

The octarepeat region of the prion protein is conformationally altered in PrP(Sc).

BACKGROUND: Prion diseases are fatal neurodegenerative disorders characterized by misfolding and aggregation of the normal prion protein PrP(C). Little is known about the details of the structural rearrangement of physiological PrP(C) into a still-elusive disease-associated conformation termed PrP(Sc). Increasing evidence suggests that the amino-terminal octapeptide sequences of PrP (huPrP, residues 59-89), though not essential, play a role in modulating prion replication and disease presentation. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that trypsin digestion of PrP(Sc) from variant and sporadic human CJD results in a disease-specific trypsin-resistant PrP(Sc) fragment including amino acids approximately 49-231, thus preserving important epitopes such as the octapeptide domain for biochemical examination. Our immunodetection analyses reveal that several epitopes buried in this region of PrP(Sc) are exposed in PrP(C). CONCLUSIONS/SIGNIFICANCE: We conclude that the octapeptide region undergoes a previously unrecognized conformational transition in the formation of PrP(Sc). This phenomenon may be relevant to the mechanism by which the amino terminus of PrP(C) participates in PrP(Sc) conversion, and may also be exploited for diagnostic purposes.

Aß40 oligomers identified as a potential biomarker for the diagnosis of Alzheimer's disease.

Alzheimer's Disease (AD) is the most prevalent form of dementia worldwide, yet the development of therapeutics has been hampered by the absence of suitable biomarkers to diagnose the disease in its early stages prior to the formation of amyloid plaques and the occurrence of irreversible neuronal damage. Since oligomeric Aß species have been implicated in the pathophysiology of AD, we reasoned that they may correlate with the onset of disease. As such, we have developed a novel misfolded protein assay for the detection of soluble oligomers composed of Aß x-40 and x-42 peptide (hereafter Aß40 and Aß42) from cerebrospinal fluid (CSF). Preliminary validation of this assay with 36 clinical samples demonstrated the presence of aggregated Aß40 in the CSF of AD patients. Together with measurements of total Aß42, diagnostic sensitivity and specificity greater than 95% and 90%, respectively, were achieved. Although larger sample populations will be needed to confirm this diagnostic sensitivity, our studies demonstrate a sensitive method of detecting circulating Aß40 oligomers from AD CSF and suggest that these oligomers could be a powerful new biomarker for the early detection of AD.