My laboratory is using cell biological techniques to examine the mechanism of prion propagation in mammalian cells, prion curing in yeast cells, and the mechanism of huntingtin (Htt) toxicity in mammalian and yeast cells. It is now understood that conversion of the properly folded prion protein, PrPc, to the misfolded amyloid conformation, PrPsc, occurs upon exposure of PrPc to misfolded PrPsc either at the plasma membrane or along its trafficking itinerary in mammalian cells. However, we still do not understand the mechanism of conversion or where in the cell conversion takes place. To determine this, we have used different Rab mutants and knocked out specific proteins to block trafficking along particular pathways. Our results show that inhibiting maturation of the MVB clears PrPsc, which indicates that the MVB is the major internal site of PrPsc conversion. The MVB is an organelle that is an intermediate in the trafficking of cargo from the early endosome to the lysosome where proteins are degraded. Interestingly, the MVB has an unusual geometry with intralumenal vesicles that contain cargo targeted for lysosomal degradation. This unusual geometry allows apposition of two sets of membranes and it may be this geometry that induces prion conversion both at the plasma membrane and in the MVB. Another project in my laboratory has been examining is the curing of the yeast PSI+ prion, which is formed from misfolded Sup35 amyloid aggregates. Similar to other yeast prions, PSI+ is cured by inactivation of Hsp104, which is required for the severing of the prion seeds. Surprisingly, PSI+ is also cured by overexpression of Hsp104 by a mechanism that is not yet understood although this process has been investigated by many different laboratories. One suggested model for curing by Hsp104 overexpression is due to asymmetric segregation of the seeds between mother and daughter cells. However, we ruled out this model by using flow cytometry to separate yeast based on age. Then by using live cell imaging of GFP-labeled Sup35, we observed that Hsp104 causes a reduction in the size of the prion seeds, suggesting that the excess Hsp104 might be removing molecules from the ends of the prion fibers. However, we find that this activity of Hsp104, which we have termed trimming, is not by itself sufficient to cure PSI+. In addition, the amyloid core that is left by the trimming activity is presented by the excess Hsp104 and other chaperones to the proteasome for degradation. This mechanism of curing by overexpression has demonstrated a new way for the cell to rid itself of prion aggregates. Finally, we have also examined the toxicity caused by Htt fragments in both yeast and mammalian cells in an attempt to understand why they cause neurodegeneration. We first found that in yeast, the toxicity of the Htt fragments with expanded polyglutamine repeats depends on the prion that is present in the yeast along with the Htt fragment. We found that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, was significantly more toxic in PSI+ yeast than in PIN+ even though HttQP103 formed multiple aggregates in both PSI+ and PIN+ yeast. Furthermore, the toxicity caused by HttQP103 aggregates was effectively rescued by expressing the soluble C-terminal fragment of Sup35. This shows that the toxicity of HttQP103 in yeast containing the PSI+ prion is primarily due to sequestration of the essential protein, Sup35. Interestingly, expression of HttQ103, which does not contain the polyproline repeat region, was toxic in both PSI+ and PIN+ yeast and poorly rescued by expressing the soluble C-terminal fragment of Sup35. This shows the complex nature of Htt toxicity, which may be caused by the sequestration of different proteins depending on the cell type. In related work in mammalian cells where we examined the aggregation of Htt fragments with different lengths polyQ repeat regions, we found by using fluorescence correlation spectroscopy that both non-pathological and pathological Htt fragments form soluble oligomers in the cytosol. Therefore, it is not the presence of soluble oligomers per se that causes toxicity, but the nature of the oligomers that determines whether they cause neuropathology, perhaps because different oligomers sequester different proteins.

Project Start
Project End
Budget Start
Budget End
Support Year
27
Fiscal Year
2013
Total Cost
$1,396,589
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
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Yim, Yang-In; Park, Bum-Chan; Yadavalli, Rajgopal et al. (2015) The multivesicular body is the major internal site of prion conversion. J Cell Sci 128:1434-43
Park, Yang-Nim; Zhao, Xiaohong; Yim, Yang-In et al. (2014) Hsp104 overexpression cures Saccharomyces cerevisiae [PSI+] by causing dissolution of the prion seeds. Eukaryot Cell 13:635-47
Olszewski, Maciej B; Chandris, Panagiotis; Park, Bum-Chan et al. (2014) Disruption of clathrin-mediated trafficking causes centrosome overduplication and senescence. Traffic 15:60-77
Beilina, Alexandria; Rudenko, Iakov N; Kaganovich, Alice et al. (2014) Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease. Proc Natl Acad Sci U S A 111:2626-31
Heymann, J Bernard; Winkler, Dennis C; Yim, Yang-In et al. (2013) Clathrin-coated vesicles from brain have small payloads: a cryo-electron tomographic study. J Struct Biol 184:43-51
Park, Yang-Nim; Morales, David; Rubinson, Emily H et al. (2012) Differences in the curing of [PSI+] prion by various methods of Hsp104 inactivation. PLoS One 7:e37692
Edvardson, Simon; Cinnamon, Yuval; Ta-Shma, Asaf et al. (2012) A deleterious mutation in DNAJC6 encoding the neuronal-specific clathrin-uncoating co-chaperone auxilin, is associated with juvenile parkinsonism. PLoS One 7:e36458
Zhao, Xiaohong; Park, Yang-Nim; Todor, Horia et al. (2012) Sequestration of Sup35 by aggregates of huntingtin fragments causes toxicity of [PSI+] yeast. J Biol Chem 287:23346-55
Park, Yang-Nim; Zhao, Xiaohong; Norton, Mark et al. (2012) Huntingtin fragments and SOD1 mutants form soluble oligomers in the cell. PLoS One 7:e40329
Park, Yang-Nim; Masison, Daniel; Eisenberg, Evan et al. (2011) Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae. Yeast 28:673-81

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