In the current study, we have demonstrated a molecular interaction between human PrP and Hsp70. Colocalization analyses show that cellular Hsp70 exactly overlaps with the cytosolic forms of PrPs in cells expressing Cyto-PrP and PG14-PrP but not wild-type PG5-PrP or ER-retained PrPs, including 3AV-PrP and ER-PrP. Furthermore, we demonstrate that activation of cellular Hsp70, by overexpression of recombinant Hsp70 or pharmacological treatment with geldanamycin, selectively promoted the degradation of and restored the relevant cytotoxicity of the cytosolic PrPs but not wild-type or ER-retained PrPs in cultured cells. These data indicate that cytosolic PrPs are an authentic substrate of Hsp70.
Hsp70 is a potent molecular chaperone that aids in the folding and refolding of nascent proteins and denatured proteins, respectively. More importantly, when a protein cannot be correctly renatured, Hsp70 can mediate degradation of the protein [17, 18]. Misfolded/mislocalized proteins in the cytoplasm are degraded via the ubiquitin-proteasome pathway. It has been repeatedly reported that the overexpression of Hsp70 facilitates the degradation of α-synuclein , a risk factor for PD, and eliminates the hyperphosphorylated microtubule-associated protein tau (tau) , a primary pathological component of AD. Therefore, activating Hsp70 is one potential strategy and therapeutic target for treating AD and PD. Similar to the abnormal protein deposits found in the brains of patients with AD and PD, the accumulation of PrPSc in the brain is a predominant neuropathological feature of prion diseases. However, numerous evidence has suggested that PrPSc is not toxic; however, based on experiments in cultured cells and transgenic mice, abnormal PrPs, e.g., Cyto-PrP  or PrPs mutated in the transmembrane region  or in the octarepeat region , have been proposed to be neurotoxic. These data highlight the importance of clearing continuously produced misfolded PrPs that might be intermediates of PrPSc. Hsp70 may work as an efficient host factor to clear toxic misfolded or mislocalized PrPs in the cytoplasm.
Our results indicate that the accumulation of cytosolic PrPs increases the levels of cellular Hsp70, which may reflect a triggered mechanism for cell defense. Additionally, our study shows that Hsp70 is significantly activated in the brains of scrapie-infected hamsters, which is consistent with what has been observed in the brain tissues of CJD patients and in animal models of TSE . Although PrPSc is more protease-resistant and cannot be degraded via the Hsp70-mediated ubiquitin-proteasome dependent pathway, several studies have demonstrated that PrPSc can translocate into the cytosol of prion-infected cells and induce toxicity [20, 21]. Up-regulating Hsp70 in cells would help clear out the penetrated PrPSc or newly formed prion intermediates.
Hsp70 is usually present in the cytoplasm and mediates the efficient modification of misfolded/mislocalized proteins in the cytoplasm. According to this study, this function is highly location specific because Hsp70 did not affect PrP proteins that retain or accumulate in ER. Correlated with the induced degradation of cytosolic PrPs, the viability of cells expressing the cytosolic PrP mutants is markedly improved. Recently, Fernandez-Funez et al. have described that the wild-type PrP can spontaneously convert into an insoluble protease-sensitive isoform in neurons of the transgenic fly brain expressing wild-type hamaster PrP, meanwhile, over-expression of Hsp70 is capable of preventing the accumulation of PrPSc-like conformers and reducing the neurotoxicity, which suggest that Hsp70 may be a therapeutic candidate for prion diseases . However, the complexes of Hsp70 and PrP is detectable in the fraction of membrane microdomains of aged flies’ brains, which seems to be distinct from the data in this study. The exact reason for this discrepancy remains unknown. Hsp70 usually presents in the cytoplasm of mammalian cells . Similarly, Hsp70 is mainly observed in the cytoplasm in the young transgenic PrP flies, but detected also in the microsome of the old flies. Probably due to the wide distribution of transgenic PrP protein in the membrane microdomains in the flies, it eventually forms a stress along with the spontaneous conversion. In that situation, Hsp70 moves across the membranous structures and into organelles. In fact, the stress-caused shift of Hsp70 from cytoplasm to membrane has been already described. In our experimental condition, the PrP constructs located mainly in membrane and organelle (ER) are transiently expressed, which may be too short to induce such “stress”. That might explain why no complex of PrP-Hsp70 is detected in the fraction of membrane/organelles. Additionally, our data also illustrate an increased Hsp70 level in the brains of scrapie strain 263K-infected hamsters. Further assays of co-localization of PrP and Hsp70 in subcellular organelles, as well as in different fractions of brain homogenates will supply more solid conclusion. Moreover, evidence suggests that the conversion of Hsp70’s function in protein-folding to its function as a degradation factor is mediated by the co-chaperone CHIP . A range of cellular agents, e.g., glucocorticoid receptor , ErbB2  and tau , are known substrates of CHIP. Additionally, DnaJ (Hsp40) is thought to link the Hsp70 chaperone machine to the ubiquitin-proteasome system . The potential contributions of CHIP and Hsp40 to the Hsp70-mediated protection against PrP-neurotoxicity will be a valuable research topic.