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• W2059821314 abstract "Sacchromyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a soluble protein that can assemble into fibers that are similar to the fibers observed in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatography, sedimentation velocity, and electron microscopy demonstrates that the soluble form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, and tetrameric form whose abundance is concentration-dependent. By the use of limited proteolysis, intrinsic fluorescence, and circular dichroism measurements, we bring strong evidence for the existence of at least two structural domains in Ure2p molecules. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insoluble high molecular weight oligomers. These changes essentially affect the COOH-terminal part of the molecule. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP. Sacchromyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a soluble protein that can assemble into fibers that are similar to the fibers observed in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatography, sedimentation velocity, and electron microscopy demonstrates that the soluble form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, and tetrameric form whose abundance is concentration-dependent. By the use of limited proteolysis, intrinsic fluorescence, and circular dichroism measurements, we bring strong evidence for the existence of at least two structural domains in Ure2p molecules. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insoluble high molecular weight oligomers. These changes essentially affect the COOH-terminal part of the molecule. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP. Keen interest has been shown during the past several years to a phenomenon of protein misfolding and aggregation (inside the cells), particularly due to studies of various amyloidosis and prion diseases (1Sunde M. Blake C. Adv. Protein Chem. 1997; 50: 123-159Crossref PubMed Google Scholar, 2Kelly J.W. Colon W. Lai Z. Lashuel H.A. McCulloch J. McCutchen S.L. Miroy G.J. Peterson S.A. Adv. Protein Chem. 1997; 50: 161-181Crossref PubMed Google Scholar, 3Horwich A.L. Weissman J.S. Cell. 1997; 89: 499-510Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar, 5Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar, 6Cohen F.E. Prusiner S.B. Annu. Rev. Biochem. 1998; 67: 793-819Crossref PubMed Scopus (472) Google Scholar). Even though it became more or less clear that conformational changes of proteins are required for the propagation of the diseases (1Sunde M. Blake C. Adv. Protein Chem. 1997; 50: 123-159Crossref PubMed Google Scholar, 2Kelly J.W. Colon W. Lai Z. Lashuel H.A. McCulloch J. McCutchen S.L. Miroy G.J. Peterson S.A. Adv. Protein Chem. 1997; 50: 161-181Crossref PubMed Google Scholar, 3Horwich A.L. Weissman J.S. Cell. 1997; 89: 499-510Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar, 5Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar, 6Cohen F.E. Prusiner S.B. Annu. Rev. Biochem. 1998; 67: 793-819Crossref PubMed Scopus (472) Google Scholar), it is not yet known how and where these conformational changes take place in vivo. There are some significant differences between prion diseases and amyloid diseases, such as transmissibility of prion diseases, but it is clear that conversion of a soluble form of a protein into insoluble aggregate is a key mechanism involved in all the cases (1Sunde M. Blake C. Adv. Protein Chem. 1997; 50: 123-159Crossref PubMed Google Scholar, 2Kelly J.W. Colon W. Lai Z. Lashuel H.A. McCulloch J. McCutchen S.L. Miroy G.J. Peterson S.A. Adv. Protein Chem. 1997; 50: 161-181Crossref PubMed Google Scholar, 3Horwich A.L. Weissman J.S. Cell. 1997; 89: 499-510Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar, 5Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar, 6Cohen F.E. Prusiner S.B. Annu. Rev. Biochem. 1998; 67: 793-819Crossref PubMed Scopus (472) Google Scholar). Such aggregates reveal high resistance to proteases, thus escaping different common degradation pathways, e.g.proteosomal complexes (7Mayer A. Siegel N.R. Schwartz A.L. Ciechanover A. Science. 1989; 244: 1480-1483Crossref PubMed Scopus (77) Google Scholar, 8Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2223) Google Scholar). These aggregates form deposits (1Sunde M. Blake C. Adv. Protein Chem. 1997; 50: 123-159Crossref PubMed Google Scholar, 2Kelly J.W. Colon W. Lai Z. Lashuel H.A. McCulloch J. McCutchen S.L. Miroy G.J. Peterson S.A. Adv. Protein Chem. 1997; 50: 161-181Crossref PubMed Google Scholar, 3Horwich A.L. Weissman J.S. Cell. 1997; 89: 499-510Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar, 5Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar, 6Cohen F.E. Prusiner S.B. Annu. Rev. Biochem. 1998; 67: 793-819Crossref PubMed Scopus (472) Google Scholar) that could lead to cytotoxicity.In contrast to mammalian prions, which in their aggregated form significantly damage the cells (leading finally to cell death), the so-called yeast prion-like proteins (Sup35p and Ure2p, when thought to be aggregated) do not damage yeast cells but do, however, change their phenotypes (9Lindquist S. Cell. 1997; 89: 495-498Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 10Wickner R.B. Annu. Rev. Genet. 1996; 30: 109-139Crossref PubMed Scopus (67) Google Scholar). This change in yeast cell phenotypes could lead to an evolutionary advantage under certain conditions, e.g.giving rise to new proteins due to a readthrough of a stop codon (in case of the aggregation of Sup35p, known to be a translation termination factor (see Refs. 9Lindquist S. Cell. 1997; 89: 495-498Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar and 11Kushnirov V.V. Ter-Avanesyan M.D. Cell. 1998; 94: 13-16Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar)), or allow the uptake of both poor and rich nitrogen sources as happens in case of the Ure2p aggregation (Ure2p is a transcriptional factor, regulator of nitrogen metabolism in yeast (12Lacroute F. J. Bacteriol. 1971; 106: 519-522Crossref PubMed Google Scholar, 13Coschigano P.W. Magasanik B. Mol. Cell. Biol. 1991; 11: 822-832Crossref PubMed Scopus (222) Google Scholar)).It is now widely accepted that formation of insoluble aggregates is a result of a shift in equilibrium between native soluble conformer of a prion protein and aggregation-competent molecules (6Cohen F.E. Prusiner S.B. Annu. Rev. Biochem. 1998; 67: 793-819Crossref PubMed Scopus (472) Google Scholar). Although reasons for such a shift are rather obscure, the basis for partitioning between different conformers seems to be provided (at least in case of mammalian prion proteins) by their specific structural properties (4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar,14Riek R. Hornemann S. Wider G. Glockshuber R. Wuthrich K. FEBS Lett. 1997; 413: 282-288Crossref PubMed Scopus (660) Google Scholar, 15Donne D.G. Viles J.H. Groth D. Mehlhorn I. James T.L. Cohen F.E. Prusiner S.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13452-13457Crossref PubMed Scopus (637) Google Scholar). Indeed, recent structural studies demonstrated that mammalian prion proteins possess a large unstructured NH2-terminal part (14Riek R. Hornemann S. Wider G. Glockshuber R. Wuthrich K. FEBS Lett. 1997; 413: 282-288Crossref PubMed Scopus (660) Google Scholar, 15Donne D.G. Viles J.H. Groth D. Mehlhorn I. James T.L. Cohen F.E. Prusiner S.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13452-13457Crossref PubMed Scopus (637) Google Scholar). Since synthetic peptides reproducing various regions in this domain are capable of polymerizing into amyloid fibrils (for review see Ref. 16Mihara H. Takahashi Y. Ueno A. Biopolymers. 1998; 47: 83-92Crossref PubMed Scopus (38) Google Scholar), this part of the molecule has been suggested to govern PrP aggregation. The most widely accepted model for amyloid formation hypothesizes that primary conformational changes affect the NH2-terminal domain of PrP and then propagate to the rest of the molecule with an efficiency depending on the local structural properties of different PrP isoforms (4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar, 6Cohen F.E. Prusiner S.B. Annu. Rev. Biochem. 1998; 67: 793-819Crossref PubMed Scopus (472) Google Scholar).In order to understand better the features that can provide the basis for possible structural plasticity of the yeast prion-like protein Ure2, we have attempted its purification and characterization (after overexpression in Escherichia coli cells). Our data demonstrate that recombinant Ure2p is a soluble monomeric protein that can self-associate into dimers, tetramers, as well as insoluble high molecular weight oligomers. These high molecular weight oligomers are fibrillar structures that appear, when examined in the electron microscope, to be very similar to the fibers that are observed in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p oligomerization is a cooperative process that is concentration-dependent. Finally, Ure2p fibers bind Congo Red as do amyloid fibers. We also bring evidence in this work for the existence of at least two structural domains in Ure2p molecules and show that only slight conformational changes accompany Ure2p aggregation. These changes affect essentially the COOH-terminal part of the molecule.DISCUSSIONYeast prions Sup35 and Ure2p do not kill the cells that harbor them and are not hazardous nor pathogenic for humans. They present, therefore, a useful model system to study the molecular mechanisms of mammalian prions advent and spread. Our capacity to express recombinant yeast prion Ure2p as a soluble protein allowed us to characterize some of its properties. The work presented here provides new insights into the structure of this protein and the mechanism of its assembly into various quaternary structures.Structure of Ure2pSoluble Ure2p was found to emerge from a sizing column with an apparent molecular mass of 130,000 incompatible with that expected for a monomeric Ure2p molecule that would have a globular shape. Sedimentation velocity measurements reveal that recombinant Ure2p forms oligomers in a concentration-dependent manner. Indeed, soluble Ure2p preparations are heterogeneous mixtures of monomeric, dimeric, and tetrameric Ure2p molecules as well as higher order oligomers. Examination of soluble Ure2p preparations by electron microscopy reveals globular but heterogeneous particles. The most striking structures we observed were ring-shaped particles with an outer diameter of 10 nm composed of four arrowheads pointed toward the center of the particles. The finding that Ure2p oligomerization is concentration-dependent indicates that the monomeric, dimeric, and tetrameric forms of Ure2p as well as the higher molecular weight oligomers are in equilibrium. Over 95% of the soluble form of Ure2p was found to assemble into insoluble high molecular weight oligomers either upon incubation of the protein for prolonged periods at 4 or 28 °C or adjustment of the pH of the solution from 7.5 to 6.5. In the latter case fibrillar structures highly heterogeneous in size and shape were obtained while fibrils that are 15–20 nm wide varying in length between 0.5 and 10 μm were obtained upon incubation of the soluble form of Ure2p at either 4 or 28 °C. These fibers appear very similar in the electron microscope to PrP scrapie-associated filaments and to the fibers observed upon Sup35 autoassembly and bind Congo Red which is a characteristic of amyloid fibers.The kinetics of Ure2p assembly are sigmoidal indicating a cooperative process. Furthermore, the lag phase preceding Ure2p assembly depends on the concentration of the protein and disappears upon addition of preformed Ure2p fibers to the soluble form of the proteins which indicates that Ure2p autoassembly is a nucleated process. Finally, the critical concentration for Ure2p assembly appears to be lower than 0.5 μm since the proportion of protein assembled into fibers represents 95% of the input soluble Ure2p (10 μm). Taken together, the properties of Ure2p in vitro can account for the propagation of Ure2p polymers in a manner similar to what occurs in the case of Sup 35 and PrP.Conformational Changes of Ure2pSeveral genetic studies suggest that Ure2p is a two-domain protein that were recently shown by means of the two-hybrid system to interact with each other (38Fernandez-Bellot E. Guillemet E. Baudin-Baillieu A. Gaumer S. Komar A.A. Cullin C. Biochem. J. 1999; 338: 403-407Crossref PubMed Scopus (27) Google Scholar). Indeed, the NH2-terminal third of the molecule appears essential for the occurrence of [URE3] phenotype, whereas the catalytic activity of the protein appears to be located in the COOH-terminal two-thirds of the molecule (10Wickner R.B. Annu. Rev. Genet. 1996; 30: 109-139Crossref PubMed Scopus (67) Google Scholar, 39Masison D.C. Maddelein M.L. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12503-12508Crossref PubMed Scopus (146) Google Scholar). Secondary structure calculation algorithms predict that most of the NH2-terminal third of the protein is unstructured (i.e. essentially constituted by coils, turns, and sheets), whereas the α-helical structures are mainly located in the COOH-terminal two-thirds of the protein. Thus Ure2p is predicted to be at least a two-domain protein. These predictions are supported by the data obtained upon treatment of the COOH- and NH2-terminal domains of the protein, expressed independently, with different proteases. Ure2p NH2-terminal domain showed a much higher sensitivity to protease treatments than the COOH-terminal domain of the protein. Indeed, none of the peptides generated upon protease treatment of the NH2-terminal domain resisted longer than 1 min, whereas the products of Ure2p COOH-terminal domain cleavage were stable for over 1 h. These studies revealed two fragments (22 and 14.5 kDa) highly resistant to proteinase K as well as two others (29 and 7 kDa) highly resistant to trypsin. These peptides were tentatively identified as a breakdown products of Ure2p COOH-terminal domain since they are generated upon treatment of Ure2p 94–354 fragment by the same proteases. The largest peptide (29 kDa) would correspond to Ure2p COOH-terminal domain devoid of its 8–11 NH2-terminal amino acid residues or to the same domain devoid of its 10 COOH-terminal amino acid residues. Confirmation of the assignment will ultimately require the isolation and sequencing of the 29-kDa fragment. The same approach will lead to assignment of the 22-, 14.5-, and 7-kDa fragments.A major conformational change occurring upon Ure2p aggregation would result in differences in the digestion profiles of the two forms of the protein. This is not what we observed. Indeed, treatment of full-length Ure2p in its soluble or aggregated forms by proteinase K or trypsin yielded peptides that are identical to that generated upon treatment of Ure2p COOH-terminal domain by the same proteases. However, the kinetics of cleavage were significantly slower for the insoluble form of Ure2p. Such results are what one expects in the case of a reduced accessibility of Ure2p (the substrate) to the protease. Furthermore, the peptides generated upon either proteinase K or trypsin treatments of full-length Ure2p were that generated upon treatment of the COOH-terminal domain of Ure2p by the same proteases. This strongly suggests that the NH2-terminal domain is rapidly degraded whether or not the COOH-terminal domain is present. The rapid and total degradation of Ure2p NH2-terminal domain would be a consequence of its lack of structure and compactness. Finally, our findings also indicate that the COOH-terminal domain adopts the same conformation in the presence or absence of the NH2-terminal domain, since the same protease cleavage sites are exposed whether the NH2-terminal domain of the protein is present or not.Proteolytic treatment of proteins is a powerful tool to probe conformational changes that may affect the structure of the substrate polypeptides. Nevertheless, a number of tiny changes may well be overlooked. In order to detect such small changes, intrinsic fluorescence as well as circular dichroism measurements were carried out. Given that all tryptophan and tyrosine residues are located within Ure2p COOH-terminal domain, intrinsic fluorescence measurements allowed us to follow conformational changes affecting this domain of the protein. The data presented in this work clearly demonstrate that the COOH-terminal domain of Ure2p undergoes a conformational change during self-assembly process that results in a 50% decrease in exposure of tyrosine and tryptophan residues to water concomitant with a shift in the wavelength of the emission maximum. Circular dichroism data suggest that a number of amino acid residues in Ure2p are in α-helical structures. The amount of α-helices increases when the protein is in the presence of TFE, a solvent known to stabilize α-helical structure. This could be due to the transition of stretches of amino acid residues in Ure2p NH2-terminal domain from random coils to α-helical structures. Alternatively, the increase of the content of α-helices could correspond to amino acid residues located within the COOH-terminal moiety of the protein that adopts a structure predominantly α-helical upon TFE addition. Consequently, methods allowing the specific labeling of the Ure2p NH2-terminal domain with extrinsic fluorophores will have to be designed in order to access the effect of the solvent as well as autoassembly on the flexibility of this domain.Present genetic, biochemical, and structural data (3Horwich A.L. Weissman J.S. Cell. 1997; 89: 499-510Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar, 5Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar, 9Lindquist S. Cell. 1997; 89: 495-498Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 10Wickner R.B. Annu. Rev. Genet. 1996; 30: 109-139Crossref PubMed Scopus (67) Google Scholar, 39Masison D.C. Maddelein M.L. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12503-12508Crossref PubMed Scopus (146) Google Scholar) allow us to make a brief comparison of some properties of mammalian prion protein PrP and yeast prions, Ure2p and Sup35p. In all cases proteins seem to consist of at least two major parts. Indeed, although the NH2-terminal part (bearing unusual amino acids repeats, rich in Gly in the case of PrP (4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar, 6Cohen F.E. Prusiner S.B. Annu. Rev. Biochem. 1998; 67: 793-819Crossref PubMed Scopus (472) Google Scholar) and in Asn and Gln in the case of Ure2p and Sup35 (10Wickner R.B. Annu. Rev. Genet. 1996; 30: 109-139Crossref PubMed Scopus (67) Google Scholar)) seems to be in all the cases unstructured and flexible and at the same time absolutely required for the propagation of prion conditions (through an extensive aggregation of proteins), the COOH-terminal part appears to be compactly folded. We show here that Ure2p like Sup35 and PrPc can undergo extensive aggregation into highly ordered fibers. Our data demonstrate that preformed Ure2p fibers incorporate the soluble form of the protein and propagate Ure2p assembly in a manner similar to what is observed in the case of Sup35 (32Glover J.R. Kowal A.S. Schirmer E.C. Patino M.M. Liu J.J. Lindquist S. Cell. 1997; 89: 811-819Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 40Paushkin S.V. Kushnirov V.V. Smirnov V.N. Ter-Avanesyan M.D. Science. 1997; 277: 381-383Crossref PubMed Scopus (198) Google Scholar) and support a “protein only” seeded polymerization model for Ure2p.Conversion of the cellular form of the prion protein (PrPc) to the scrapie isoform (PrPSc) (leading further to its aggregation) is thought to be driven by an α-helical to β-sheet conformational transition (4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar, 5Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar). The poorly structured NH2-terminal part of the prion protein (14Riek R. Hornemann S. Wider G. Glockshuber R. Wuthrich K. FEBS Lett. 1997; 413: 282-288Crossref PubMed Scopus (660) Google Scholar, 15Donne D.G. Viles J.H. Groth D. Mehlhorn I. James T.L. Cohen F.E. Prusiner S.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13452-13457Crossref PubMed Scopus (637) Google Scholar) seems to be ultimately crucial in such α → β interconversion, providing the plasticity required for a conformational change. This characteristic is common among PrPc, Sup35, as well as Ure2p. Nevertheless, such conformational changes are not necessarily restricted to the NH2-terminal part of these proteins. Indeed, the majority of the mutations affecting the efficiency of propagation of prion diseases is associated with the COOH-terminal part of the protein (4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar, 5Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar). Our finding that the conformation of the COOH-terminal part of Ure2p is affected during aggregation is in favor of a mechanism of assembly similar among all prions. Keen interest has been shown during the past several years to a phenomenon of protein misfolding and aggregation (inside the cells), particularly due to studies of various amyloidosis and prion diseases (1Sunde M. Blake C. Adv. Protein Chem. 1997; 50: 123-159Crossref PubMed Google Scholar, 2Kelly J.W. Colon W. Lai Z. Lashuel H.A. McCulloch J. McCutchen S.L. Miroy G.J. Peterson S.A. Adv. Protein Chem. 1997; 50: 161-181Crossref PubMed Google Scholar, 3Horwich A.L. Weissman J.S. Cell. 1997; 89: 499-510Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar, 5Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar, 6Cohen F.E. Prusiner S.B. Annu. Rev. Biochem. 1998; 67: 793-819Crossref PubMed Scopus (472) Google Scholar). Even though it became more or less clear that conformational changes of proteins are required for the propagation of the diseases (1Sunde M. Blake C. Adv. Protein Chem. 1997; 50: 123-159Crossref PubMed Google Scholar, 2Kelly J.W. Colon W. Lai Z. Lashuel H.A. McCulloch J. McCutchen S.L. Miroy G.J. Peterson S.A. Adv. Protein Chem. 1997; 50: 161-181Crossref PubMed Google Scholar, 3Horwich A.L. Weissman J.S. Cell. 1997; 89: 499-510Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar, 5Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar, 6Cohen F.E. Prusiner S.B. Annu. Rev. Biochem. 1998; 67: 793-819Crossref PubMed Scopus (472) Google Scholar), it is not yet known how and where these conformational changes take place in vivo. There are some significant differences between prion diseases and amyloid diseases, such as transmissibility of prion diseases, but it is clear that conversion of a soluble form of a protein into insoluble aggregate is a key mechanism involved in all the cases (1Sunde M. Blake C. Adv. Protein Chem. 1997; 50: 123-159Crossref PubMed Google Scholar, 2Kelly J.W. Colon W. Lai Z. Lashuel H.A. McCulloch J. McCutchen S.L. Miroy G.J. Peterson S.A. Adv. Protein Chem. 1997; 50: 161-181Crossref PubMed Google Scholar, 3Horwich A.L. Weissman J.S. Cell. 1997; 89: 499-510Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar, 5Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar, 6Cohen F.E. Prusiner S.B. Annu. Rev. Biochem. 1998; 67: 793-819Crossref PubMed Scopus (472) Google Scholar). Such aggregates reveal high resistance to proteases, thus escaping different common degradation pathways, e.g.proteosomal complexes (7Mayer A. Siegel N.R. Schwartz A.L. Ciechanover A. Science. 1989; 244: 1480-1483Crossref PubMed Scopus (77) Google Scholar, 8Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2223) Google Scholar). These aggregates form deposits (1Sunde M. Blake C. Adv. Protein Chem. 1997; 50: 123-159Crossref PubMed Google Scholar, 2Kelly J.W. Colon W. Lai Z. Lashuel H.A. McCulloch J. McCutchen S.L. Miroy G.J. Peterson S.A. Adv. Protein Chem. 1997; 50: 161-181Crossref PubMed Google Scholar, 3Horwich A.L. Weissman J.S. Cell. 1997; 89: 499-510Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar, 5Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar, 6Cohen F.E. Prusiner S.B. Annu. Rev. Biochem. 1998; 67: 793-819Crossref PubMed Scopus (472) Google Scholar) that could lead to cytotoxicity. In contrast to mammalian prions, which in their aggregated form significantly damage the cells (leading finally to cell death), the so-called yeast prion-like proteins (Sup35p and Ure2p, when thought to be aggregated) do not damage yeast cells but do, however, change their phenotypes (9Lindquist S. Cell. 1997; 89: 495-498Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 10Wickner R.B. Annu. Rev. Genet. 1996; 30: 109-139Crossref PubMed Scopus (67) Google Scholar). This change in yeast cell phenotypes could lead to an evolutionary advantage under certain conditions, e.g.giving rise to new proteins due to a readthrough of a stop codon (in case of the aggregation of Sup35p, known to be a translation termination factor (see Refs. 9Lindquist S. Cell. 1997; 89: 495-498Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar and 11Kushnirov V.V. Ter-Avanesyan M.D. Cell. 1998; 94: 13-16Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar)), or allow the uptake of both poor and rich nitrogen sources as happens in case of the Ure2p aggregation (Ure2p is a transcriptional factor, regulator of nitrogen metabolism in yeast (12Lacroute F. J. Bacteriol. 1971; 106: 519-522Crossref PubMed Google Scholar, 13Coschigano P.W. Magasanik B. Mol. Cell. Biol. 1991; 11: 822-832Crossref PubMed Scopus (222) Google Scholar)). It is now widely accepted that formation of insoluble aggregates is a result of a shift in equilibrium between native soluble conformer of a prion protein and aggregation-competent molecules (6Cohen F.E. Prusiner S.B. Annu. Rev. Biochem. 1998; 67: 793-819Crossref PubMed Scopus (472) Google Scholar). Although reasons for such a shift are rather obscure, the basis for partitioning between different conformers seems to be provided (at least in case of mammalian prion proteins) by their specific structural properties (4Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar,14Riek R. Hornemann S. Wider G. Glockshuber R. Wuthrich K. FEBS Lett. 1997; 413: 282-288Crossref PubMed Scopus (660) Google Scholar, 15Donne D.G. Viles J.H. Groth D. Mehlhorn I. James T.L. Cohen F.E. Prusiner S.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13452-13457Crossref PubMed Scopus (637) Google Scholar). Indeed, recent structural studies demonstrated that mammalian prion proteins possess a large unstr" @default.
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