Dynamics Of In Vitro Amyloid Fiber Aggregation And Cytotoxicity Of Yeast Prion Sup35NM And Its Variants

Numerous human diseases are linked to the formation of amyloid fibrils, includingAlzheimer's disease, Parkinson's disease, and the transmissible spongiformencephalopathies (TSEs). Although the proteins associated with amyloid diseaseshave diverse amino acid sequences, the amyloid fibers that they form seem to share acommon structure. Amyloid fibers are highly organized protein aggregatescharacterized by filamentous morphology, highβ-sheet content, protease resistance,and yellow-green birefringence upon staining with Congo red. Investigations for themechanism by which amyloid forms will no doubt provide not only the clues for theunderstanding of amyloid diseases but also the development of strategies of prevetionand treatment to such diseases. Howerver, despite considerable studies had been done,much is still unknown about the relationship between amino acid sequence andpropensity to form amyloid.In 1994, Wickner et al proposed that two non-Mendelian elements, [URE3] and[PSI~+], are prion forms of the Sacchromyces cerevisiae proteins, respectively. Since inboth PrP and yeast prions, prion formation is resulted from the conversion of nativeprotein into a self-propagating and infectious amyloid form, yeast prions provide auseful model for studying not only the mechanism of amyloid fibril formation but alsoprion-like transmission of the protein conformation. Sup35p is an essential subunit ofthe translation termination factor, which corresponds to eRF3 in mammals. It functions in termination in a complex by binding its partner Sup45p. Overexpressionof Sup35p induces protein aggregation and causes [PSI~-] cells to revert to [PSI~+].Sup35 is composed of three distinct sequence elements: N region, M region and Cregion. The N region (aa 1-123) is required for the maintenance of [PSI~+] and rich inQ/N (44%). The C region provides the termination function. The M region is highlycharged and its function is still unclear. In order to examine the role of unusual aminoacid composition and specific sequence in the prion domain and to provide clues forthe pathogenesis of amyloid diseases, in the present study, we constructed severalSup35NM variants and tested the ability and dynamics of amyloid fiber formation ofeach variant as well as their toxicity to mammalian cells.1. Dynamics of in vitro amyloid fiber formation of Sup35NM and its variantsIn order to prove the impacts of amino acid sequence on fiber formation, weprepared 5 variants of Sup35p prion domain (N) in which the order of the amino acid2-123 was randomized while without changing the amino acids composition. Thesescrambled variants were named as Sup35NM-1, -2, -3, -4, and -5, respectively. Thewild type (wt) Sup35NM and its variants which were expressed in E. coli and purifiedunder denaturing conditions respectively were subjected to the investigations on thedynamics of amyloid fibers formation. The morphological alteration of the wtSup35NM and variants in PBS (pH 7.4) during the protein aggregation was visualizedby transmission electron microscopy (TEM). The results revealed that at 2h after theinitiation of the assembly reaction, wt Sup35NM and its variants showed a mixture ofgranule, oligomer, and short filaments. After 18h, only fibers could be observed insamples of NM-1,-2, -3 and wt NM, while NM-4, -5 exhibited a mixture of structuresincluding fibers and oligomers. After 48h, all samples exhibited only fibers. Thecircular dichroism (CD) assay showed that the course of amyloid fiber formationunderwent a conformational shift fromα-helix toβ-sheet. The amyloid form wasdetectable when the fibers of either wt Sup35NM or its variants were treated with 1μg/ml or 4μg/ml (final concentration) protease K at 37℃for 60 min. The fibersshowed higher protease K resistance when compared with the native forms. ThTfluorescence assay displayed a rapid growth and a final equilibrium phase in wtSup35NM, Sup35NM-1, -2, and -3. However, the ThT fluorescence curve ofSup35NM-4, and -5 revealed a distinct decrease in conversion kinetics with aprolonged lag time of nearly 3～5 hours. The results showed that the aggregation rateof Sup35NM-4, -5 is much slower than the others. As monomeric form of wtSup35NM and its variants decreased, insoluble polymers increased gradually asmonitored by SDS-PAGE and Western blot. After the fibers were heated in boiledwater bath, ThT fluorescence intensity decreased significantly and monomers of allsamples reappeared in SDS-PAGE. The results indicated that the randomized priondomains polymerized into amyloid in vitro under native conditions, however, theaggregating speed of these variants is somewhat different. Moreover, the assembledand ordered filaments retain all the morphological, structural and biochemicalproperties reported for ex vivo Sup35p aggregate. In conclusion, the specific sequencefeatures can modulate the rate of conversion of Sup35p into amyloid in vitro, thoughit is dispensable to the ability of amyloid formation.2. Cross-seeding of wt Sup35NM and its variantsIt has been reported that Rnql, polyQ aggregates facilitated [PSI] appearance invivo, and Rnql, polyQ fibers enhance the in vitro formation of fibers formed bySup35NM. To explore the role of the heterologous Q/N-rich aggregates in fiberformation, we studied the reciprocal effect of wt Sup35NM and its variants onconformation conversion. The results showed that the rates of Sup35NM-1, -4, and -5conversion were increased when sonicated wt Sup35NM fibers were added, and viceversa. In addition, the other Q/N-rich amyloid, Ure2p, weakly augmented fiberformation of wt Sup35NM and its variants. Heterologous amyloid is much lesssufficient than its own conversion. On the contrary, non-Q/N-rich amyloid, α-synuclein did not seed conversion of wt Sup35NM and its variants. The resultssuggested that heterologous Q/N-rich amyloids promote the conversion of similarQ/N-rich domain.3. Toxicity of the amyloid aggregates formed by wt Sup35NM and its variants tomammalian cellsA number of data have confirmed that pre-fibrillar aggregates of proteins are toxicto cells. It has not been clarified whether the aggregates of Sup35p are still toxic tomammalian cells. In the initial of this study, incubation of wt Sup35NM and itsvariants (64μmol/L) for 0.5～1h at room temperature or 2d～4d at 4℃, led to theformation of early aggregates and mature fibers, respectively. Then, Vero, NIH-3T3,and SH-SY5Y cells were incubated in the aliquots of solutions containing differentmorphological types of aggregates with 5, 10, 15μmol/L final concentrations for 24h.The results revealed that cells exposed to early aggregates of wt Sup35NM and itsvariants displayed significantly reduced viability with the increase of aggregatesconcentrations, whereas mature fibrils are harmless. These results are similar to thatof other proteins reported previously, implying that the cytotoxicity isstructure-dependent. However, the cell types exposed to early aggregates alsodisplayed variable susceptibility to damage. Vero, NIH-3T3 cells are susceptible,while SH-SY5Y cells are resistant, indicating that the cytotoxicity is archived in aspecific way. Moreover, another phenomenon was notable that when Vero, NIH-3T3cells were incubated with wt Sup35NM and its variants fibers after heating in boiledwater bath for 20min, these cells expressed the similar damage just as the earlyaggregates, suggesting the structure similarity between the deaggregated maturefibrils after heating and the early aggregates.