Different pathogenic PrP mutations linked to inherited prion diseases in humans interfere with the maturation of PrP in the secretory pathway (review in Tatzelt and Winklhofer 2004). The ER lumen is known to contain quality control systems to prevent further transit of aberrant polypeptides (review in Ellgaard and Helenius 2003; McCracken and Brodsky 2003). Misfolded proteins in the ER can be degraded through a mechanism known as ER-associated protein degradation (ERAD). This pathway involves recognition of misfolded polypeptides by chaperones, retrograde transport into the cytosol, and subsequent proteasomal degradation (Finley et al. 1984; Hurtley and Helenius 1989; Jentsch et al. 1987; Klausner and Sitia 1990).
Several groups observed degradation of wild type and mutant PrP via the proteasomal pathway, which was interpreted as evidence for retrograde transport of PrP from the ER lumen into the cytosol (Jin et al. 2000; Ma and Lindquist 2001; Yedidia et al. 2001; Zanusso et al. 1999). This conclusion, however, was discussed controversially after cytosolic accumulation of PrP was proposed to be an experimental artifact, due to the prolonged treatment of cells with proteasomal inhibitors and overexpression of PrP driven by a viral promotor (Drisaldi et al. 2003). Similarly, we did not observe that significant fractions of the two pathogenic PrP mutants T183A and F198S were subjected to proteasomal degradation (Kiachopoulos et al. 2005). Moreover, misfolded PrP in the ER seems not to activate the unfolded protein response (UPR) pathway. Induction of the UPR leads to the upregulation of a variety of proteins, such as the ER chaperone BiP (Ma and Hendershot 2001; Patil and Walter 2001). However, none of the misfolded PrP mutants analyzed induces increased transcription of BiP in a detectable manner (Winklhofer et al. 2003c).
A possible role of Grp94 on the maturation of PrPC emerged from studies on a defective stress response in scrapie-infected N2a cells (Tatzelt et al. 1995; Winklhofer et al. 2001). Grp94 is among the most abundant ER proteins, yet its physiological function is largely unknown (review in Argon and Simen 1999). When we treated cultured cells with geldanamycin, we observed the stabilization of a high mannose glycoform of PrPC (Winklhofer et al. 2003a), an observation corroborated later (Ochel et al. 2003). Similarly to inhibitors of a-mannosidases, geldanamycin prevents the processing of high mannose glycoforms into complex structures. Trafficking of immature PrPC to the outer leaflet of the plasma membrane is not impaired by geldanamycin. Moreover, in scrapie-infected cells high mannose glycoforms of PrPC are preferred substrates for the formation of PrPSc (Winklhofer et al. 2003a). Geldanamycin, a benzochinone ansamycin derivative, has been shown to interact with Hsp90 in vitro and in vivo and to interfere with Hsp90 functions (review in Neckers 2002). Later on, geldanamycin was also reported to bind Grp94, the ER homolog of Hsp90 (Chavany et al. 1996). Since PrPC has no cytoplasmic domain, we assumed that Grp94 might be involved in the geldanamycin-induced effects on glycosylation. Geldanamycin might either prolong a pre-existing interaction of immature PrPC with Grp94 or impair a Grp94 function, which is required for mannose trimming (Winklhofer et al. 2003a). Interestingly, a transient interaction of high mannose glycoform of PrPC and Grp94 was described in the absence of geldanamycin (Capellari et al. 1999).
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