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Biological Chemistry

Detecting Misfolded Protein Aggregates

Biochemistry: Assay might aid early disease diagnosis

by Laura Cassiday
April 29, 2011 | A version of this story appeared in Volume 89, Issue 18

Credit: Nephron/Wikipedia
In Alzheimer’s disease, amyloid- proteins misfold and form soluble aggregates, which accumulate over time into plaques (brown) that are on average tens of micrometers wide.
Credit: Nephron/Wikipedia
In Alzheimer’s disease, amyloid- proteins misfold and form soluble aggregates, which accumulate over time into plaques (brown) that are on average tens of micrometers wide.

A new assay detects small, soluble aggregates of misfolded proteins that form at early stages of diseases—such as Alzheimer’s, Parkinson’s, and type 2 diabetes. The technique could enable earlier diagnosis than is now possible, as well as enhance scientists’ understanding of misfolded protein disorders, say the researchers who developed the tool.

These aggregates form when proteins, such as amyloid-β in Alzheimer’s disease or α-synuclein in Parkinson’s, don’t fold correctly and then stack together to form structures known as cross-β sheets. If enough proteins accumulate, they produce insoluble bundles called amyloid fibrils.

Although scientists once blamed amyloid fibrils for disease development, many now believe that these large protein aggregates form relatively late in the course of disease. “Over the past 10 years, it’s become apparent that smaller, soluble aggregates are actually more toxic than the amyloid deposits,” says Joseph P. Fedynyshyn, a scientist at Novartis Vaccines & Diagnostics, in Emeryville, Calif. The small aggregates likely form before a patient experiences symptoms, he says.

At late stages of disease, pathologists can use special dyes to stain amyloid fibers in tissue samples. But doctors have had no way to detect smaller, soluble aggregates at early stages of disease, when therapies are more likely to be effective. Fedynyshyn, Cleo M. Salisbury, and their colleagues now have developed a reagent that recognizes a negatively charged region that is a common feature in many misfolded protein aggregates (Biochemistry, DOI: 10.1021/bi200215j).

The team had previously discovered that a positively charged peptide derived from a human prion—a protein that, when misfolded, causes Creutzfeldt-Jakob disease—interacts with the misfolded form of the prion but not with the properly folded form. To see whether the peptide also interacts with other misfolded proteins, the researchers modified the peptide’s backbone to increase its stability and then attached it to paramagnetic beads. They found that a variety of misfolded proteins, including α-synuclein and amyloid-β, could bind to these bead-peptide complexes, which the researchers isolated with a magnet. They removed the bound proteins from the beads and detected them with specific antibodies. The investigators think that the positively charged peptide binds to the negatively charged portion of the misfolded protein aggregates.

The researchers next tested whether their peptide-coated beads could detect aggregates in a more clinical context. They added various sizes of misfolded amyloid-β aggregates to human cerebrospinal fluid and used the beads to purify the aggregates. The method was sensitive enough to detect all aggregate sizes, including the smallest amyloid-β aggregate tested, which contained 12 to 16 protein monomers and is toxic to neurons in patients with Alzheimer’s disease.

The researchers have already used the technique to detect misfolded protein aggregates in cerebrospinal fluid from Alzheimer’s patients (PLoS One, DOI:10.1371/journal.pone.0015725). They now plan to test the diagnostic usefulness of the assay in larger groups of patients with Alzhei­mer’s and other diseases.

Adriano Aguzzi, a neuropathologist at the University Hospital of Zurich, predicts that the reagent will prove useful for diagnosing multiple misfolded protein diseases. He also says that because this peptide interacts with many types of aggregates, the “study highlights the similarities among prions and other protein-misfolding diseases.”


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