Prion protein (PrP) adopts either a helical conformation (PrP^C) or an alternative, beta sheet-rich, misfolded conformation (PrP^Sc). The PrP^Sc form has the ability to “infect” PrP^C and force it into the misfolded state. Accumulation of PrP^Sc is associated with a number of lethal neurodegenerative disorders, including Creutzfeldt-Jacob disease (CJD). Knockout of PrP^C protects cells and animals from PrP^Sc infection; thus, there is interest in identifying factors that regulate PrP^C stability, with the therapeutic goal of reducing PrP^C levels and limiting infection by PrP^Sc. Here, we assembled a short-hairpin RNA (shRNA) library composed of 25+ shRNA sequences for each of 133 protein homeostasis (aka proteostasis) factors, such as molecular chaperones and co-chaperones. This Proteostasis shRNA Library was used to identify regulators of PrP^C stability in HEK293 Hu129M cells. Strikingly, the screen identified a number of Hsp70 family members and their co-chaperones as putative targets. Indeed, a chemical pan-inhibitor of Hsp70s reduced PrP^C levels and limited conversion to PrP^Sc in N2a cells. These results implicate specific proteostasis sub-networks, especially the Hsp70 system, as potential new targets for the treatment of CJD. More broadly, the Proteostasis shRNA Library might be a useful tool for asking which proteostasis factors are important for a given protein.

朊病毒蛋白 (PrP) 采用螺旋构象 (PrP ^C ) 或另一种富含 β 折叠的错误折叠构象 (PrP ^Sc )。 PrP ^Sc形式具有“感染”PrP ^C并迫使其进入错误折叠状态的能力。 PrP ^Sc的积累与许多致命的神经退行性疾病有关，包括克雅氏病 (CJD)。 PrP ^C的敲除可保护细胞和动物免受 PrP ^Sc感染；因此，人们有兴趣确定调节 PrP ^C稳定性的因素，其治疗目标是降低 PrP ^C水平并限制 PrP ^Sc的感染。在这里，我们组装了一个短发夹 RNA (shRNA) 文库，该文库由 133 个蛋白质稳态（又称蛋白质稳态）因子（例如分子伴侣和共伴侣）中的每一个的 25 个以上 shRNA 序列组成。该蛋白质稳态 shRNA 文库用于鉴定 HEK293 Hu129M 细胞中 PrP ^C稳定性的调节因子。引人注目的是，屏幕将一些 Hsp70 家族成员及其共同伴侣确定为假定目标。事实上，Hsp70 的化学泛抑制剂降低了 N2a 细胞中 PrP ^C水平并限制了向 PrP ^Sc的转化。这些结果表明特定的蛋白质稳态子网络，尤其是 Hsp70 系统，是治疗克雅氏病的潜在新靶点。更广泛地说，蛋白质稳态 shRNA 文库可能是一个有用的工具，用于询问哪些蛋白质稳态因子对给定蛋白质很重要。