No abstract

No abstract

In recent studies, the amyloid fibrils produced in vitro from recombinant prion protein encompassing residues 89-230 (rPrP 89-230) were shown to produce transmissible form of prion disease in transgenic mice (Legname et al., (2004) Science 305, 673-676). Long incubation time observed upon inoculation of the amyloid fibrils, however, suggests that the fibrils generated in vitro have low infectivity titers. These results emphasize the need to define optimal conditions for prion conversion in vitro, under which high levels of infectivity can be generated in a cell-free system. Because copper(II) has been implicated in normal and pathological functions of the prion protein, here we investigated the effect of Cu(2+) on cell-free conversion of recombinant PrP. Our results show that at pH 7.2 and at micromolar concentrations, Cu(2+) inhibited conversion of full-length recombinant PrP (rPrP 23-230) into amyloid fibrils. This effect was most pronounced for Cu(2+), and less so for Zn(2+), while Mn(2+) had no effect on the conversion. Cu(2+)-dependent inhibition of the amyloid formation was less effective at pH 6.0, at which rPrP 23-230 displays lower Cu(2+)-binding capacity. Using rPrP 89-230, we found that Cu(2+)-dependent inhibition occurred even in the absence of octarepeat region; however, it was less effective. Our further studies indicated that Cu(2+) inhibited conversion by stabilizing a nonamyloidogenic PK-resistant form of alpha-rPrP. Remarkably, Cu(2+) also had a profound effect on preformed amyloid fibrils. When added to the fibrils, Cu(2+) induced long-range coiling of individual fibrils and enhanced their PK-resistance. It, however, produced only minor changes in their secondary structures. In addition, Cu(2+) induced further aggregation of the amyloid fibrils into large clumps, presumably, through interfibrillar coordination of copper ions by octarepeats. Taken together, our studies suggest that the role of Cu(2+) in the pathogenesis of prion diseases is complex. Because Cu(2+) may inhibit prion replication, while at the same time stabilize disease-specific isoform against proteolytic clearance, the final outcome of copper-induced effect on progression of prion disease may not be straightforward.

No abstract

No abstract

In recent studies, the amyloid form of recombinant prion protein (PrP) encompassing residues 89-230 (rPrP 89-230) produced in vitro induced transmissible prion disease in mice. These studies showed that unlike "classical" PrP Sc produced in vivo, the amyloid fibrils generated in vitro were more proteinase-K sensitive. Here we demonstrate that the amyloid form contains a proteinase K-resistant core composed only of residues 152/153-230 and 162-230. The PK-resistant fragments of the amyloid form are similar to those observed upon PK digestion of a minor subpopulation of PrP Sc recently identified in patients with sporadic Creutzfeldt-Jakob disease (CJD). Remarkably, this core is sufficient for self-propagating activity in vitro and preserves a ␤-sheet-rich fibrillar structure. Full-length recombinant PrP 23-230, however, generates two subpopulations of amyloid in vitro: One is similar to the minor subpopulation of PrP Sc , and the other to classical PrP Sc . Since no cellular factors or templates were used for generation of the amyloid fibrils in vitro, we speculate that formation of the subpopulation of PrP Sc with a short PK-resistant C-terminal region reflects an intrinsic property of PrP rather than the influence of cellular environments and/or cofactors. Our work significantly increases our understanding of the biochemical nature of prion infectious agents and provides a fundamental insight into the mechanisms of prions biogenesis.Keywords: prion protein; amyloid fibrils; conformational transition; proteinase K; Creutzfeldt-Jakob disease Several severe neurodegenerative diseases including Creutzfeldt-Jakob disease (CJD), Gerstmann-StrausslerSheinker disease, and fatal familial insomnia are associated with misfolding and aggregation of the prion protein (Prusiner 2001). Prion maladies manifest themselves in infectious, familial, and sporadic forms. To explain the infectious form of the prion diseases, the "protein-only hypothesis" postulates that an abnormal isoform of the prion protein, PrP Sc , acts as an infectious agent and propagates its pathological conformation in an autocatalytic manner using the normal isoform of the same protein, PrP C , as a substrate (Prusiner 1982). PrP C and PrP Sc differ substantially in their conformations. Unlike PrP C , PrP Sc is a multimeric assembly characterized by enhanced resistance to proteinase K (PK) digestion and by an increase in the amount of ␤-sheet structure Reprint requests to: Ilia V. Baskakov, 725 W. Lombard Street, Baltimore, MD 21201, USA; e-mail: Baskakov@umbi.umd.edu; fax: (410) 706-8184.Abbreviations: CJD, Creutzfeldt-Jakob disease; spCJD, sporadic CJD; PrP, prion protein; PrP C , the normal, cellular isoform of PrP; PrP Sc , the abnormal, infections isoform of PrP; rPrP, recombinant PrP; rPrP 89-230, recombinant PrP encompassing residues 89-230; rPrP 23-230, full-length recombinant PrP; ␣-rPrP 89-230, ␣-helical isoform of rPrP 89-231; ␣-rPrP 23-230, ␣-helical isoform of rPrP 23-231; PK, proteinase K; PrP 27-30, PK-resistant core of classical PrP ...

Flax (Linum usitatissimum L.) phloem fibers elongate considerably during their development and intrude between existing cells. We questioned whether fiber elongation is caused by cell tip growth or intercalary growth. Cells with tip growth are characterized by having two specific zones of cytoplasm in the cell tip, one with vesicles and no large organelles at the very tip and one with various organelles amongst others longitudinally arranged cortical microtubules in the subapex. Such zones were not observed in elongating flax fibers. Instead, organelles moved into the very tip region, and cortical microtubules showed transversal and helical configurations as known for cells growing in intercalary way. In addition, pulse-chase experiments with Calcofluor White resulted in a spotted fluorescence in the cell wall all over the length of the fiber. Therefore, it is concluded that fiber elongation is not achieved by tip growth but by intercalary growth. The intrusively growing fiber is a coenocytic cell that has no plasmodesmata, making the fibers a symplastically isolated domain within the stem.

In recent studies, we developed a protocol for in vitro conversion of full-length mouse recombinant PrP (Mo rPrP23-230) into amyloid fibrils [Bocharova et al. (2005) J. Mol. Biol. 346, 645-659]. Because amyloid fibrils produced from recombinant Mo PrP89-230 display infectivity [Legname et al. (2004) Science 305, 673-676], polymerizatiom of rPrPs in vitro represents a valuable model for elucidating the mechanism of prion conversion. Unexpectedly, when the same conversion protocol was used for hamster (Ha) rPrP23-231, we experienced substantial difficulties in forming fibrils. While searching for potential reasons of our failure to produce fibrils, we probed the effect of methionine oxidation in rPrP. We found that oxidation of methionines interferes with the formation of rPrP fibrils and that this effect is more profound for Ha than for Mo rPrP. To minimize the level of spontaneous oxidation, we developed a new protocol for rPrP purification, in which highly amyloidogenic Ha rPrP with minimal levels of oxidized residues was produced. Furthermore, our studies revealed that oxidation of methionines in preformed fibrils inhibited subsequent maturation of fibrils into proteinase K-resistant PrP(Sc)-like conformation (PrP-res). Our data are consistent with the proposition that conformational changes within the central region of the protein (residues 90-140) are essential for adopting PrP-res conformation and demonstrate that methionine oxidation interferes with this process. These studies provide new insight into the mechanism of prion polymerization, solve a long-standing practical problem in producing PrP-res fibrils from full-length PrP, and may help in identifying new genetic and environmental factors that modulate prion disease.