Abstract
BackgroundMutations in the PRNP gene account for ~15% of all prion disease cases. Little is understood about the mechanism of how some of these mutations in PRNP cause the protein to aggregate into amyloid fibers or cause disease. We have taken advantage of a chimeric protein system to study the oligopeptide repeat domain (ORD) expansions of the prion protein, PrP, and their effect on protein aggregation and amyloid fiber formation. We replaced the ORD of the yeast prion protein Sup35p with that from wild type and expanded ORDs of PrP and compared their biochemical properties in vitro. We previously determined that these chimeric proteins maintain the [PSI+] yeast prion phenotype in vivo. Interestingly, we noted that the repeat expanded chimeric prions seemed to be able to maintain a stronger strain of [PSI+] and convert from [psi-] to [PSI+] with a much higher frequency. In this study we have attempted to understand the biochemical properties of these chimeric proteins and to establish a system to study the properties of the ORD of PrP both in vivo and in vitro.ResultsInvestigation of the chimeric proteins in vitro reveals that repeat-expansions increase aggregation propensity and that the kinetics of fiber formation depends on the number of repeats. The fiber formation reactions are promiscuous in that the chimeric protein containing 14 repeats can readily cross-seed fiber formation of proteins that have the wild type number of repeats. Morphologically, the amyloid fibers formed by repeat-expanded proteins associate with each other to form large clumps that were not as prevalent in fibers formed by proteins containing the wild type number of repeats. Despite the increased aggregation propensity and lateral association of the repeat expanded proteins, there was no corresponding increase in the stability of the fibers formed. Therefore, we predict that the differences in fibers formed with different repeat lengths may not be due to gross changes in the amyloid core.ConclusionThe biochemical observations presented here explain the properties of these chimeric proteins previously observed in yeast. More importantly, they suggest a mechanism for the observed correlation between age of onset and disease severity with respect to the length of the ORD in humans.
Highlights
IntroductionLittle is understood about the mechanism of how some of these mutations in PRNP cause the protein to aggregate into amyloid fibers or cause disease
Mutations in the PRNP gene account for ~15% of all prion disease cases
They suggest a mechanism for the observed correlation between age of onset and disease severity with respect to the length of the oligopeptide repeat domain (ORD) in humans
Summary
Little is understood about the mechanism of how some of these mutations in PRNP cause the protein to aggregate into amyloid fibers or cause disease. The prion protein (PrP) can misfold and aggregate to form amyloid fibers, leading to neurodegenerative prion diseases [3,4]. The three primary means of acquiring prion diseases are as follows: 1) infection – can be transmitted by the ingestion of meat obtained from diseased animals, 2) spontaneous – occurs sporadically via unknown mechanism(s) and 3) inherited – mutations in the PRNP gene encoding PrP [4,5]. We use a model system created to investigate some aspects of an inherited form of prion disease that results from insertional mutations in PRNP to cause diseases such as GSS and CJD [9]
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