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The addition of the hydrophobic-hydrophilic 19 amino acids stretch to the N-terminus of firefly luciferase also increased the frequency of aggregate formation.
Other chaperones such as HSP70 or HSPB8 have also been shown to reduce the frequency of aggregate formation in vitro [55], [57].
The frequency of aggregate formation was similar for the two extended Mca1 versions (Mca1454 and Mca1451) and also for the truncation mutants of the amino-terminal hydrophobic-hydrophilic stretch, Mca1mut1 (containing the hydrophobic part) and Mca1mut2 (containing the hydrophilic part).
As displayed in Figure 3D, the frequency of aggregate formation was not altered by the different truncations, indicating that the presence of either hydrophilic or hydrophobic stretch at the N-terminus of the prion-forming domain of Mca1 is sufficient to destabilize the protein conformation.
A quantitative analysis of the frequency of aggregate formation in cells deleted for chromosomal MCA1 revealed Mca1454-GFP and Mca1451-GFP aggregates in about 50% of the cells, whereas we found Mca1432-GFP aggregates in only about 10% of the analyzed cells (Figure 3B).
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The scoring index used in this study to judge the presence of platelet aggregates in the blood smear was limited in that only the size, but not the frequency of aggregates, was investigated.
Intermittent stirring regimes led to a higher frequency of aggregates over the size of 200 µm in the Solohill Collagen cultures (Fig. 2C i) and D i)), while this was much less pronounced in the Cultispher-S cultures.
However, it was not possible to assess whether raising PSA levels had similar protective effects in cells expressing WT huntingtin exon 1 GFP due to their very low frequency of aggregates and cell death.
We additionally analyzed the effects of Mca1454 GFP aggregation in cells containing a wild type copy of MCA1 and found no difference in the frequency of GFP aggregate formation (Figure 3C).
However, the frequency of GFP aggregates was 3-fold increased for FFL19aa-GFP although the aggregation was clearly less pronounced compared to Mca1451 (Figure 3D).
The model accurately decribes the influence of both energy dissipation rate and protein concentration on the initial breakage frequency of the aggregates.
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