Our laboratory is interested in the formation and dissolution of both normal and pathological protein complexes in the cell with an emphasis on the role of molecular chaperones in these processes. We have continued our studies on the role of Hsc70 in clathrin-mediated endocytosis by generating knockout mice of the HSC70 cochaperones, neuronal-specific auxilin and ubiquitously expressed GAK. Specifically, we wanted to determine the effect of knocking out GAK and also determine the domains of GAK that are essential for its cochaperone function. Using mouse embryonic fibroblasts (MEFs) derived from the conditional GAK knockout mouse, GAK was disrupted by using adenovirus expressing Cre recombinase. Knocking-out GAK, completely blocked clathrin-mediated endocytosis in the MEFs and disrupted the clathrin organization on the plasma membrane and at the trans-Golgi network. Moreover, the GAK-depleted MEFs were arrested at the G1/S checkpoint, but still underwent multiple rounds of centrosome duplication. Interestingly, all of these phenotypes were rescued with a truncated GAK molecule consisting of just the clathrin binding domain and J-domain. Furthermore, this truncated GAK fragment, which was expressed as a transgene, was able to rescue the lethality observed when GAK was knocked out of specific tissues in mice. These results show that the clathrin-binding and J-domains of GAK, but not the kinase and pTen-like domains of GAK, are essential for its cochaperone function. In addition, we also studied the propagation of prions, infective proteins that can misfold into an amyloid conformation both in mammalian and yeast cells. In mammalian cells, chronically infected scrapie cells, having the misfolded amyloid form of prion, have been shown to be cured by a number of different drugs that have been reported to have different target sites. However, immunostaining of scrapie showed that with all these different anti-prion drugs, the scrapie localized was no longer found cycling throughout the endosomal trafficking pathway, but instead was all localized in lamp-1 positive endosomes. These results indicate that the mechanism by which these drugs clear scrapie is to sequester scrapie in a dead-end compartment, which, in turn, prevents the conversion of the properly folded prion protein to the misfolded amyloid conformation. Our research on prion has also been extended to yeast, which is a simple model system to study prion propagation. In yeast, the molecular chaperone, Hsp104, regulates the inheritance of several yeast prions including PSI+, which is the prion form of the translation termination factor Sup35p. Both inactivation and overexpression of Hsp104 cures yeast of the PSI plus prion. By using live cell imaging, we have now found that the molecular chaperone, Hsp104, has two activities. It not only severs prion seeds, but also trims or reduces the size of the seeds by removing Sup35 molecules from the end of the amyloid fiber. These released Sup35 molecules do not nucleate new seeds and therefore, trimming does not increase the number of prion seeds. In contrast, severing the does seeds increase the number of new seeds. We have also now found that trimming activity is important for curing of plus prion by overexpression of Hsp104, as well as maintaining the steady-state size of the prion seeds in propagating PSI plus yeast.

Project Start
Project End
Budget Start
Budget End
Support Year
25
Fiscal Year
2011
Total Cost
$1,913,748
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
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Zip Code
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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