Many neurodegenerative diseases are caused by the accumulation of intracellular or extracellular proteins that form amyloid deposits. In general, amyloid deposits can cross cell membranes to spread the toxic amyloids to neighboring cells. Since this mode of propagation is similar to the seeding that occurs in infectious prion disease, prion-like transmission appears to be common to many neurodegenerative diseases. To protect against the accumulation of toxic amyloid aggregates, molecular chaperones function to disaggregate them, but when they do not maintain quality control, protein aggregates are formed. My research is at the crossroads of these two areas;examining the formation and propagation of protein aggregates and their disaggregation by molecular chaperones in yeast and mammalian cells. Yeast prions require the molecular chaperone, Hsp104, which propagates prion in dividing yeast by severing the prion seeds. Yeast prions are cured that is rid of the amyloid propagating conformation, by inactivation of Hsp104, but paradoxically, one of the yeast prions, PSI+, is cured by overexpression of Hsp104. By using live cell imaging of GFP-labeled Sup35, which forms PSI+ prion, we found that curing by Hsp104 overexpression was actually caused by an activity of Hsp104 that we recently uncovered termed trimming. Trimming reduces the size of the seeds by removing monomers from the ends of the amyloid seeds without creating new seeds, in contrast to the severing activity of Hsp104 that creates new seeds In a second study in yeast, we examined the basis of huntingtin (Htt) toxicity. HttpolyQ fragments form toxic aggregates only in yeast propagating a prion. We found that depending on the prion, different essential proteins are sequestered by the Htt aggregates. In addition the amino acid composition of the Htt fragment also affected which proteins are sequestered. Future work will include overexpressing different Hsp104 fragments and also Hsp104 from different yeast species to better understand the mechanism of PSI+ curing by Hsp104 overexpression. The ability of Hsp104 to depolymerize amyloids has potential as a therapeutic tool to treat amyloid diseases in man. Interestingly, overexpression of Htt has also been found to cure a prion, in this case URE3, and we are investigating the mechanism of the curing. We have also studied both protein aggregation and the role of molecular chaperones in protein disaggregation in mammalian cells. Our long-term study of the role of the molecular chaperone, Hsc70, in clathrin-dependent trafficking has shown that the major function of Hsc70 is to prevent aggregation of clathrin in the cytosol. We discovered that this activity is dependent on two J-domain proteins that interact with Hsc70 and clathrin, the ubiquitously expressed GAK and the neuronal-specific auxilin. These J-domain proteins are essential for all clathrin-dependent trafficking in the cell and, when these proteins were knocked in the mouse, this caused both developmental and neurological defects. By expressing fragments of GAK, we found its pten-like domain of GAK is not essential, which is surprising, since it was proposed that this domain is required for irreversible uncoating of clathrin coated vesicles. Furthermore, we found that there was an association between this domain and Parkinsons disease, an area that we are now pursuing by using our GAK and auxilin knockout mice. Another major research project carried out was studying prion propagation in mammalian cells. The conversion of the PrPc to the amyloid PrPsc has been shown to occur as PrPc traffics along the endosomal pathway, but the compartment where conversion takes place has been controversial. By blocking the cellular trafficking of PrPsc at various points, we found that the intracellular site of prion conversion is the multivesicular body (MVB). This observation is of particular interest because the MVB has an unusual membrane topology that might enable trans-interaction between PrPc and PrPsc molecules within the cell as well as at the plasma membrane. We are now perturbing other trafficking pathways both to confirm our results and to understand how other aspects of trafficking (e.g. clathrin-mediated endocytosis) affect PrPsc propagation.

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28
Fiscal Year
2014
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U.S. National Heart Lung and Blood Inst
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Greene, Lois E; Zhao, Xiaohong; Eisenberg, Evan (2018) Curing of [PSI+] by Hsp104 Overexpression: Clues to solving the puzzle. Prion 12:9-15
Zhao, Xiaohong; Lanz, Jenna; Steinberg, Danielle et al. (2018) Real-time imaging of yeast cells reveals several distinct mechanisms of curing of the [URE3] prion. J Biol Chem 293:3104-3117
Zhao, Xiaohong; Rodriguez, Ramon; Silberman, Rebecca E et al. (2017) Heat shock protein 104 (Hsp104)-mediated curing of [PSI(+)] yeast prions depends on both [PSI(+)] conformation and the properties of the Hsp104 homologs. J Biol Chem 292:8630-8641
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
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
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
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

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