Autocatalytic cleavage of lithostathine leads to the formation of quadruple-helical fibrils (QHF-litho) that are present in Alzheimer's disease. Here we show that such fibrils also occur in Creutzfeldt-Jakob and Gerstmann-Sträussler-Scheinker diseases, where they form protease-K-resistant deposits and co-localize with amyloid plaques formed from prion protein. Lithostathine does not appear to change its native-like, globular structure during fibril formation. However, we obtained evidence that a cluster of six conserved tryptophans, positioned around a surface loop, could act as a mobile structural element that can be swapped between adjacent protein molecules, thereby enabling the formation of higher order fibril bundles. Despite their association with these clinical amyloid deposits, QHF-litho differ from typical amyloid fibrils in several ways, for example they produce a different infrared spectrum and cannot bind Congo Red, suggesting that they may not represent amyloid structures themselves. Instead, we suggest that lithostathine constitutes a novel component decorating disease-associated amyloid fibrils. Interestingly, [6,6′]bibenzothiazolyl-2,2′-diamine, an agent found previously to disrupt aggregates of huntingtin associated with Huntington's disease, can dissociate lithostathine bundles into individual protofilaments. Disrupting QHF-litho fibrils could therefore represent a novel therapeutic strategy to combat clinical amyloidoses. Autocatalytic cleavage of lithostathine leads to the formation of quadruple-helical fibrils (QHF-litho) that are present in Alzheimer's disease. Here we show that such fibrils also occur in Creutzfeldt-Jakob and Gerstmann-Sträussler-Scheinker diseases, where they form protease-K-resistant deposits and co-localize with amyloid plaques formed from prion protein. Lithostathine does not appear to change its native-like, globular structure during fibril formation. However, we obtained evidence that a cluster of six conserved tryptophans, positioned around a surface loop, could act as a mobile structural element that can be swapped between adjacent protein molecules, thereby enabling the formation of higher order fibril bundles. Despite their association with these clinical amyloid deposits, QHF-litho differ from typical amyloid fibrils in several ways, for example they produce a different infrared spectrum and cannot bind Congo Red, suggesting that they may not represent amyloid structures themselves. Instead, we suggest that lithostathine constitutes a novel component decorating disease-associated amyloid fibrils. Interestingly, [6,6′]bibenzothiazolyl-2,2′-diamine, an agent found previously to disrupt aggregates of huntingtin associated with Huntington's disease, can dissociate lithostathine bundles into individual protofilaments. Disrupting QHF-litho fibrils could therefore represent a novel therapeutic strategy to combat clinical amyloidoses. The pathological hallmarks of many neurodegenerative diseases are fibrillar deposits of proteins and polypeptides in brain. Many of these deposits are characterized by histochemical staining properties similar to those of starch and hence were called amyloid structures. Although the causal relationship between fibrillar deposits and pathogenesis has not yet been established in all cases, there is increasing evidence that these fibrils can exert a deleterious effect. Their clinical relevance has been shown for instance in cataract formation (1Sandilands A. Hutcheson A.M. Long H.A. Prescott A.R. Vrensen G. Loster J. Klopp N. Lutz R.B. Graw J. Masaki S. Dobson C.M. MacPhee C.E. Quinlan R.A. EMBO J. 2002; 21: 6005-6014Crossref PubMed Scopus (127) Google Scholar) and in familial encephalopathy with neuroserpin inclusions (2Davis R.L. Shrimpton A.E. Holohan P.D. Bradshaw C. Feiglin D. Collins G.H. Sonderegger P. Kinter J. Becker L.M. Lacbawan F. Krasnewich D. Muenke M. Lawrence D.A. Yerby M.S. Shaw C.M. Gooptu B. Elliott P.R. Finch J.T. Carrell R.W. Lomas D.A. Nature. 1999; 401: 376-379Crossref PubMed Google Scholar); also, it is highly likely in early onset Parkinson's disease (3Conway K.A. Harper J.D. Lansbury Jr., P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1270) Google Scholar). In addition, there are some recurring correlations between fibrillar pathologies and disease-associated mutations, as in some familial systemic amyloidoses, Huntington's disease, and early onset Parkinson's disease (4Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3342-3344Crossref PubMed Scopus (514) Google Scholar). These correlations strongly suggest that fibril formation initiates the pathological events and that it is not an unavoidable epiphenomenon. In addition, the early aggregates of disease-associated polypeptides have been shown to be cytotoxic (5Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2163) Google Scholar, 6Walsh D.M. Klyubin I. Fadeeva J.V. Cullen W.K. Anwyl R. Wolfe M.S. Rowan M.J. Selkoe D.J. Nature. 2002; 416: 535-539Crossref PubMed Scopus (3719) Google Scholar). Understanding the process of fibril formation and the means by which amyloid deposits can be cleared in vivo therefore represent two major scientific challenges. Among proteins that form clinical fibril deposits, we have been working on lithostathine. We have studied its physical characteristics and observed that it readily polymerizes into fibrils after self-proteolysis of its N-terminal undecapeptide (7Cerini C. Peyrot V. Garnier C. Duplan L. Veesler S. Le Caer J.P. Bernard J.P. Bouteille H. Michel R. Vazi A. Dupuy P. Michel B. Berland Y. Verdier J.M. J. Biol. Chem. 1999; 274: 22266-22274Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). On the basis of the x-ray structure of the monomer (8Bertrand J.A. Pignol D. Bernard J.P. Verdier J.M. Dagorn J.C. Fontecilla-Camps J.C. EMBO J. 1996; 15: 2678-2684Crossref PubMed Scopus (79) Google Scholar) and high resolution electron microscopy, we showed that fibrils formed a quadruple-helical filament (QHF-litho 1The abbreviations used are: QHF-lithoquadruple-helical filaments of lithostathineCJDCreutzfeldt-Jakob diseaseCRCongo RedFTIRFourier-transformed infrared spectroscopyMALDI-TOFmatrix-assisted laser desorption ionization time-of-flight spectrometryPGL-034[6,6′]bibenzothiazolyl-2,2′-diamine.; see Ref. 9Gregoire C. Marco S. Thimonier J. Duplan L. Laurine E. Chauvin J.P. Michel B. Peyrot V. Verdier J.M. EMBO J. 2001; 20: 3313-3321Crossref PubMed Scopus (35) Google Scholar). Interestingly, these fibrils were found to be present in the pathological lesions of Alzheimer's disease (senile plaques and neurofibrillary tangles), and it is overexpressed during the very early stages of the disease before clinical signs appear (10Duplan L. Michel B. Boucraut J. Barthellemy S. Desplat-Jego S. Marin V. Gambarelli D. Bernard D. Berthezene P. Alescio-Lautier B. Verdier J.M. Neurobiol. Aging. 2001; 22: 79-88Crossref PubMed Scopus (44) Google Scholar). We therefore believe that lithostathine constitutes an important protein to investigate to understand the deposition of polypeptides in vivo in relation to neurodegenerative diseases. It has remained unclear, however, whether stacking involves conformational changes comparable with those occurring during PrPsc (prion "scrapie") formation (11Baldwin M.A. Pan K.M. Nguyen J. Huang Z. Groth D. Serban A. Gasset M. Mehlhorn I. Fletterick R.J. Cohen F.E. et al.Philos. Trans. R Soc. Lond. B Biol. Sci. 1994; 343: 435-441Crossref PubMed Scopus (37) Google Scholar), whether these fibrils are amyloid in nature, and how fibrils stack together to produce large fibers. In this paper, we have used Fourier-transform infrared spectroscopy, circular dichroism, optical absorbance, mass spectrometry, atomic force microscopy, molecular modeling studies, and immunohistochemistry to explore the structural characteristics of lithostathine fibrils, the formation of large fibers, and strategies by which this process can be disturbed. In particular, we have been interested in their potential structural and clinical relation to amyloid diseases, most notably Creutzfeldt-Jakob disease (CJD). quadruple-helical filaments of lithostathine Creutzfeldt-Jakob disease Congo Red Fourier-transformed infrared spectroscopy matrix-assisted laser desorption ionization time-of-flight spectrometry [6,6′]bibenzothiazolyl-2,2′-diamine. Recombinant Lithostathine—Lithostathine was prepared as described previously (7Cerini C. Peyrot V. Garnier C. Duplan L. Veesler S. Le Caer J.P. Bernard J.P. Bouteille H. Michel R. Vazi A. Dupuy P. Michel B. Berland Y. Verdier J.M. J. Biol. Chem. 1999; 274: 22266-22274Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In brief, recombinant lithostathine was produced in Chinese hamster ovary cells and purified on an immunoaffinity column. Lithostathine samples were then frozen in liquid nitrogen and stored at -80 °C until required. For experiments concerning fibril formation, 100 μg of lithostathine (1 mg/ml) was incubated in phosphate-buffered saline at 37 °C for 2 weeks. After centrifugation for 5 min at 13000 × g the supernatant was discarded. The pellet was then washed twice with water and resuspended in 100 μl of water, frozen in liquid nitrogen, and stored at -80 °C until required. Congo Red Binding—The capacity of lithostathine fibrils and of amyloid fibrils formed from glucagon to bind Congo Red (CR) was assessed by mixing 50-μl aliquots of solutions containing amyloid fibrils from glucagon with 450 μl of a 20 μm solution of the dye in 1 m Tris-Cl at pH 7.4. The final polypeptide concentrations were as indicated in the plot. The stock solution containing glucagons amyloid fibrils was prepared by incubating glucagon at a concentration of 1 mg/ml in 50 mm sodium phosphate, pH 2.5, at room temperature for 2 weeks. Lithostathine fibrils were prepared from stock solution (1 mg/ml) as described above. The absorption measurements were carried out at room temperature in a Cary 3 UV-visible spectrophotometer (Varian Inc., Palo Alto, CA). Fourier Transform Infrared Spectroscopy (FTIR) Experiments—FTIR spectra were recorded on a Bruker IFS28 spectrometer equipped with a liquid nitrogen-cooled MTC detector (Bruker Optics Inc., Billerica, MA). The spectra (1000-2000 scans) were recorded at a spectral resolution of 4 cm-1 and were analyzed using the OPUS/IR2 program (Bruker). To compare soluble and fibrillar lithostathine, spectra were recorded with dry samples. For this purpose, samples were deposited on to fluorine plates, and the solvent was allowed to evaporate. Spectroscopic Studies—Baseline-corrected absorbance spectra in the range of 275 to 300 nm were recorded at 37 °C with a Cary 3E spectrophotometer (Varian Inc.) characterized by a high spectral reproducibility (S.D. < 0.02 nm). Data acquisition was in steps of 0.1 nm with an acquisition time of 1 s per data point. The instrument was equipped with a home-built double sample compartment, thermostated with a Haake F3-Q circulating water bath (Thermo Haake, Karlsruhe, Germany). Protein incubation was performed in a 1-ml, 1-cm light path quartz cuvette under the following conditions: 0.47 mg/ml lithostathine in 0.5 m Tris·Cl, pH 8. The fourth derivatives of the UV spectra were evaluated with the optimized spectral shift method as described previously (12Lange R. Frank J. Saldana J.L. Balny C. Eur. Biophys. J. 1996; 24: 277-283Google Scholar) with an automated transform program designed in the laboratory. MALDI-TOF Mass Spectrometry—MALDI-TOF analyses were performed using a Voyager DE-STR mass spectrometer (PerSeptive Biosystems, Framingham, MA). The instrument was calibrated using superoxide dismutase (22951) and cytochrome c (12361) at 4 × 10-11m and 10-9m, respectively. An α-cyano-4-hydroxy-cinnamic acid matrix solution was prepared as a saturated solution in 50% acetonitrile and 0.1% trifluoroacetic acid. 0.5-μl aliquots of a lithostathine solution (1.2 mg/ml) in 20 mm phosphate buffer, pH 8, were deposited directly on the sample plate. 0.5-μl aliquots of the matrix solution were then pipetted onto each drop, which was then allowed to dry. Positive-ion mass spectra were collected in reflectron mode at an acceleration voltage of 25 kV and an extraction delay time of 350 ns. Raw data were analyzed and processed using Grams software (PerSeptive Biosystems). Electronic Microscopy Studies—A solution of lithostathine (50 ng/μl) in 100 mm Tris·Cl, pH 7.5, was mixed with a trypsin solution (2.5 ng/ml) and left for 5 min at room temperature, and [6,6′]bibenzothiazolyl-2,2′-diamine (PGL-034; Merck KgaA, Darmstadt, Germany) was then added (1 μl in 100% Me2SO, final concentration 20 μm). The mixture was incubated at 37 °C for 16 h. Samples were then stained successively with 1% uranyl acetate for 1 min. The molar ratio of lithostathine/PGL-034 was 3/20, slightly more than used previously for EM analysis of PGL-034 with hungtingtin (1.5/20; see Ref. 13Heiser V. Engemann S. Brocker W. Dunkel I. Boeddrich A. Waelter S. Nordhoff E. Lurz R. Schugardt N. Rautenberg S. Herhaus C. Barnickel G. Bottcher H. Lehrach H. Wanker E.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16400-16406Crossref PubMed Scopus (196) Google Scholar). The final preparation was then viewed on a Philips CM100 EM (FEI Company, Hillsboro, OR). Immunohistochemistry—Brain slices (8 μm) were collected onto pre-treated glass slides (Super Frost® Plus; Menzel-Glaser, Braunscheig, Germany) and baked over 1 week at 57 °C. The slides were then dewaxed and used for immunochemistry. Endogenous peroxidase activity was inhibited with 1% H2O2 in Tris-buffered saline for 30 min at room temperature. Slides were then further incubated for 30 min in 20% goat serum, 0.3% Triton in Tris-buffered saline to block nonspecific antigenic sites. Specific polyclonal antibodies to lithostathine (Romeo; see Ref. 10Duplan L. Michel B. Boucraut J. Barthellemy S. Desplat-Jego S. Marin V. Gambarelli D. Bernard D. Berthezene P. Alescio-Lautier B. Verdier J.M. Neurobiol. Aging. 2001; 22: 79-88Crossref PubMed Scopus (44) Google Scholar) were then added at 1/100 for 6 days at 4 °C. To reveal the presence of PrPsc or resistant lithostathine deposits, some slides were submitted to drastic pre-treatment before addition of antibodies. These treatments involved a combination of chemical, physical, and enzymatic methods (14Bons N. Lehmann S. Nishida N. Mestre-Frances N. Dormont D. Belli P. Delacourte A. Grassi J. Brown P. C. R. Acad. Sci. (Paris). 2002; 325: 67-74Google Scholar) used to get rid off all the PrPc. In brief, they were incubated in 0.1% trypsin solution (Merck, Darmstadt, Germany) for 5 min at 37 °C, 120 min at 121 °C in water, and finally in proteinase K (25 μg/ml; Merck) for 20 min at 37 °C. The presence of PrPsc was checked by incubating the mouse monoclonal antibody 8G8, at 1/2000, overnight at 4 °C. This antibody recognizes the human 95-110 PrP sequence. The VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA) was used for detection, using diaminobenzidine (Sigma-Aldrich) in simple staining procedures, and the VIP kit (Vector Laboratories) was used for double staining experiments. Sequence Alignment and Molecular Modeling—Multiple sequence alignment was carried out using the ClustalX program (15Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35619) Google Scholar) and manually improved. Swapped loop sequences of a set of C-type lectin proteins and prion protein were selected from the Protein Data Bank and aligned with the lithostathine sequence. Homology modeling of the lithostathine dimer was performed largely according to the principles outlined by Greer (16Greer J. Methods Enzymol. 1991; 202: 239-252Crossref PubMed Scopus (78) Google Scholar) and as previously described in detail (17Laurine E. Lafitte D. Gregoire C. Seree E. Loret E. Douillard S. Michel B. Briand C. Verdier J.M. J. Biol. Chem. 2003; Google Scholar), using the software modules InsightII, Homology, and Discover from Accelrys (San Diego, CA) and a Silicon Graphics O2 work station (SGI, Mountain View, CA). The model of the lithostathine dimer connected by a swapped loop was built from the crystal Protein Data Bank structure of flavocetin-A (1C3A; see Ref. 18Fukuda K. Mizuno H. Atoda H. Morita T. Biochemistry. 2000; 39: 1915-1923Crossref PubMed Scopus (96) Google Scholar) from habu snake venom, a coagulation factor IX-binding protein heterodimer. The lithostathine dimer was then optimized with the constant valence force field. Energy minimization was carried out with the conjugate gradient algorithm, down to a maximum derivative of 0.001 kcal/Å. Lithostathine Fibrils Did Not Possess the Classical Properties of Amyloid Fibrils—To establish whether lithostatine fibrils possess the generic amyloid structure, we tested for the presence of cross-β conformation using apple green birefringence upon staining with CR and x-ray diffraction. In the light of conflicting methods used for detecting amyloid in clinical diagnosis, such as dyes of low specificity, it has been agreed by the community that in particular CR green birefringence should be an essential part of the clinical detection of amyloid structures (19Westermark P. Benson M.D. Buxbaum J.N. Cohen A.S. Frangione B. Ikeda S. Masters C.L. Merlini G. Saraiva M.J. Sipe J.D. Amyloid. 2002; 9: 197-200Crossref PubMed Scopus (183) Google Scholar). Interestingly, although the morphology of lithostathine fibrils is very similar to that of many amyloid fibrils (9Gregoire C. Marco S. Thimonier J. Duplan L. Laurine E. Chauvin J.P. Michel B. Peyrot V. Verdier J.M. EMBO J. 2001; 20: 3313-3321Crossref PubMed Scopus (35) Google Scholar), they do not possess green birefringent properties. In fact, QHF-litho failed to even bind CR, and the dye was washed off readily from samples prepared from fibril pellets after centrifugation. The same result was obtained when we examined the CR absorption spectra in the presence of QHF-litho or glucagon fibrils that have been shown previously (20Glenner G.G. Eanes E.D. Bladen H.A. Linke R.P. Termine J.D. J. Histochem. Cytochem. 1974; 22: 1141-1158Crossref PubMed Scopus (235) Google Scholar) to be amyloid in nature. Whereas glucagon fibrils characteristically increased the CR absorption signal at 540 nm, no such effect could be detected for QHF-litho (Fig. 1A). The spectrum of QHF-litho mixed with CR had the same shape as the one recorded on the dye alone. However, a small offset value in the baseline of the two spectra was evident, presumably arising from light scattering induced by the fibrils. Consistent with this observation, we could not obtain evidence for the presence of a cross-β structure when we examined the fibril pellet with x-ray diffraction (data not shown), and also FTIR spectroscopy could not reveal aggregated β-sheet structure in QHF-litho. The sheets of amyloid or prion fibrils appear in the infrared spectrum with an amide I maximum close to 1620 cm-1 (11Baldwin M.A. Pan K.M. Nguyen J. Huang Z. Groth D. Serban A. Gasset M. Mehlhorn I. Fletterick R.J. Cohen F.E. et al.Philos. Trans. R Soc. Lond. B Biol. Sci. 1994; 343: 435-441Crossref PubMed Scopus (37) Google Scholar, 21Dong A. Prestrelski S.J. Allison S.D. Carpenter J.F. J. Pharm. Sci. 1995; 84: 415-424Abstract Full Text PDF PubMed Scopus (452) Google Scholar, 22Fink A.L. Folding Des. 1998; 3: R9-23Abstract Full Text Full Text PDF PubMed Scopus (1053) Google Scholar). In the case of QHF-litho, no such maximum could be discerned. Moreover, fibrils and the soluble protein monomers produced almost the same amide I region with a main component centered at 1650 cm-1 (Fig. 1B). These data indicate that both states of the protein contain high levels of α-helical conformation (23Haris P.I. Chapman D. Trends Biochem. Sci. 1992; 17: 328-333Abstract Full Text PDF PubMed Scopus (199) Google Scholar) and that the C-terminal part of lithostathine did not undergo substantial structural transitions similar to those known to occur during amyloid fibril formation. Taken together, we conclude that if QHF-litho fibrils were amyloid structures they would represent an entirely novel and different type of amyloid fibrils, in particular one that lacks many of the properties that have so far been considered by the community to be essential for the definition of amyloid structures (19Westermark P. Benson M.D. Buxbaum J.N. Cohen A.S. Frangione B. Ikeda S. Masters C.L. Merlini G. Saraiva M.J. Sipe J.D. Amyloid. 2002; 9: 197-200Crossref PubMed Scopus (183) Google Scholar). Instead, we suggest that QHF-litho fibrils rather represent the interesting case of a disease-associated, abnormal protein fibril that is not amyloid in nature. Modification of the Environment of Tryptophan Residues Examined by UV and Fluorescence Spectroscopy—We tested, in addition, whether the formation of fibrils by lithostathine involves a more subtle structural reorganization of the protein (that maintains its secondary structure composition). Ultraviolet absorbance in the fourth derivative mode was used as an intrinsic probe for both tryptophan and tyrosine residues. This technique allows one to enhance the generally low resolution of zero-order UV absorbance spectra and to obtain information about structural changes in the local environment of tyrosine and tryptophan residues (12Lange R. Frank J. Saldana J.L. Balny C. Eur. Biophys. J. 1996; 24: 277-283Google Scholar, 24Lange R. Bec N. Mozhaev V.V. Frank J. Eur. Biophys. J. 1996; 24: 284-292Crossref Scopus (60) Google Scholar). The fourth derivative UV absorbance spectra were characterized by two maxima, reflecting mainly the tyrosine environment at 284 nm and the tryptophan environment at 290.5 nm (Fig. 2). Under conditions where fibril formation occurs, i.e. incubation of lithostathine at 37 °C up to 4 days, no substantial change in the tyrosine derivative band was observed. However, the maximum of the tryptophan derivative band shifted from 290.7 to 291.8 nm, accompanied by an increase of the intensity of absorption. This significant red shift can be explained either by a decreased interaction of tryptophan residues with water molecules or by an increased interaction with other hydrophobic or aromatic residues. To distinguish between these two hypotheses we used fluorescence as an intrinsic probe of the solvent exposure of tryptophan residues. The initial fluorescence spectrum of lithostathine (t = 0) was characterized by an emission maximum at 340 nm, indicating that most of the tryptophan residues are exposed to water. This finding is in good agreement with our structural data (8Bertrand J.A. Pignol D. Bernard J.P. Verdier J.M. Dagorn J.C. Fontecilla-Camps J.C. EMBO J. 1996; 15: 2678-2684Crossref PubMed Scopus (79) Google Scholar). During fibril formation, however, the maximum emission wavelength did not change significantly, suggesting that the mean exposure of tryptophans to water is not affected. However, a 25% quenching of tryptophan fluorescence was observed during incubation, indicative of conformational rearrangements in the vicinity of tryptophan residues. Altogether, these results suggested that the shift in the absorption maximum and fluorescence quenching of tryptophan residues were the result of an increased interaction with hydrophobic or aromatic residues, which could occur during fibril formation. Dimerization of Lithostathine Preceded or Was Concomitant to Cleavage—To understand the process of fibril formation and, hence, the mode of interaction between the protein molecules within the fibril, we have examined the early events in fibril formation using MALDI-TOF mass spectrometry. As reported previously (7Cerini C. Peyrot V. Garnier C. Duplan L. Veesler S. Le Caer J.P. Bernard J.P. Bouteille H. Michel R. Vazi A. Dupuy P. Michel B. Berland Y. Verdier J.M. J. Biol. Chem. 1999; 274: 22266-22274Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), fibril formation depends on the self-cleavage of lithostathine, resulting in the release of an N-terminal undecapeptide from the C-terminal protein domain, which represents the precursor of lithostathine fibrils. The mass spectrum recorded at t = 0 (Fig. 3A), in the 1000 to 25000-Da mass range, contained a single charge state series at mass/charge (m/z) values of 16612.8, 8307.1, and 5545.0 that corresponded to the ionic states +1 to +3 of uncleaved lithostathine (S2). Note that the dispersion in the mass/charge ratio that can be observed in each charge state corresponds to the salt adducts. By contrast, the spectrum taken after 1 day (Fig. 3B) demonstrates the presence of uncleaved lithostathine, as well as substantial amounts of cleaved lithostathine (S1), which can be detected at m/z values of 15012.2, 7510.0, and 5000.98 (charge states +1to +3). Bearing in mind that dimerization is an essential step in fibril formation (9Gregoire C. Marco S. Thimonier J. Duplan L. Laurine E. Chauvin J.P. Michel B. Peyrot V. Verdier J.M. EMBO J. 2001; 20: 3313-3321Crossref PubMed Scopus (35) Google Scholar), we also recorded spectra in the 1000 to 45000-Da mass range. After 1 day, we found a peak at 30108.3 m/z, corresponding to the mass of S1-dimers, along with a second peak at 33488.1 m/z, representing dimers of intact lithostathine (Fig. 3B, inset). Although MALDI-TOF does not allow rigorous relative quantification of the S1 and S2 dimers, it is clear that the cleaved species disappears more rapidly than full-length lithostathine (after 3 days incubation; see Fig. 3, C (10 days) and D). This observation suggests that, as soon as it is cleaved, lithostathine assembles into multimers. In addition, the intensity of the peak corresponding to the S1 dimer is always low. Taken together, these data suggest that the S1 dimer represents only a transient species on the route to the formation of higher mass multimers. This finding is different from the one obtained for dimers containing the full-length protein that were previously shown to be unable to give rise to fibrils (9Gregoire C. Marco S. Thimonier J. Duplan L. Laurine E. Chauvin J.P. Michel B. Peyrot V. Verdier J.M. EMBO J. 2001; 20: 3313-3321Crossref PubMed Scopus (35) Google Scholar). MALDI mass spectrometry thus reveals a complex equilibrium taking place after 1 day between intact and cleaved lithostathine, in both monomeric and dimeric forms. Interestingly, traces of S2 dimers are already present at t = 0, which leads us to conclude that lithostathine dimerization either precedes or is concomitant with its cleavage. By contrast, the presence of an uncleaved N-terminal undecapeptide blocks the assembly of the dimers into fibrils of high order. Lithostathine Is a Member of a Protein Family That Is Pre-disposed to Undergo Domain Swapping—The results described above showed that the formation of lithostathine fibrils does not significantly perturb the secondary structure of the protein but could involve rearrangements of the local environment of the tryptophan residues. A possible explanation of these effects has emerged from sequence analysis and homology modeling. Previously, a sequence segment encompassing part of the tryptophan cluster (residues Trp89-Gly105) was found to be involved in the formation of protein-protein contacts during fibril formation (9Gregoire C. Marco S. Thimonier J. Duplan L. Laurine E. Chauvin J.P. Michel B. Peyrot V. Verdier J.M. EMBO J. 2001; 20: 3313-3321Crossref PubMed Scopus (35) Google Scholar). Lithostathine belongs to the C-type lectin superfamily (25Drickamer K. J. Biol. Chem. 1988; 263: 9557-9560Abstract Full Text PDF PubMed Google Scholar). By multiple sequence alignment of lithostathine with other C-type lectins able to swap, we found that the sequence of this segment is conserved, in particular with respect to the tryptophan residues (Fig. 4). For reasons outlined in more detail below, this region will hereafter be referred to as the loop region, and it is flanked by sequences that contain, at least in lithostathine, several proline and glycine residues forming hinge regions. Both amino acids were shown previously (26Bergdoll M. Remy M.H. Cagnon C. Masson J.M. Dumas P. Structure. 1997; 5: 391-401Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 27Ramoni R. Vincent F. Ashcroft A.E. Accornero P. Grolli S. Valencia C. Tegoni M. Cambillau C. Biochem. J. 2002; 365: 739-748Crossref PubMed Google Scholar) to define the hinge region of proteins that possess mobile structural elements. Hence, we tentatively termed these two sequence segments the N- and C-terminal hinge regions as used previously in C-type lectins from snake venoms (18Fukuda K. Mizuno H. Atoda H. Morita T. Biochemistry. 2000; 39: 1915-1923Crossref PubMed Scopus (96) Google Scholar). When we examined the structure and position of the loop region in the known structures of C-type lectins we noted that flavocetin-A is a disulfide-linked dimer (α and β chains) (Protein Data Bank number 1C3A; see Ref. 18Fukuda K. Mizuno H. Atoda H. Morita T. Biochemistry. 2000; 39: 1915-1923Crossref PubMed Scopus (96) Google Scholar) consisting of two different but homologous subunits that interact via a swapped and extended loop region. The root mean square deviation of the two subunits compared with lithostathine is 2.7 and 3.3 Å for the α and β chain, respectively. The major difference occurred within the loop region (Fig. 5A). By contrast, a backbone superimposition of the loop regions of the flavocetin-A β chain and lithostathine resulted in an root mean square deviation of only 0.75 Å (Fig. 5B), demonstrating that the local conformation of these regions is clearly similar. Taken together, these data raise the possibility that lithostathine has the capacity to undergo a domain swapping reaction in
By two-dimensional polyacrylamide gel electrophoresis of 30S ribosomal subunit proteins (S proteins) from Haloarcula marismortui we identified 27 distinct spots and analyzed all of them by protein sequence analysis. We demonstrated that protein HmaS2 (HS2) is encoded by the open reading frame orfMSG and has sequence similarities to the S2 ribosomal protein family. The proteins HmaS5 and HmaS14 were identified as spots HS7 and HS21/HS22, respectively. Protein HS4 was characterized by amino-terminal sequence analysis. The spot HS25 was recognized as an individual protein and also characterized by sequence analysis. Furthermore, the complete primary sequence of HS26 is reported, showing similarity only to eukaryotic ribosomal proteins. The sequence data of a further basic protein shows a high degree of similarity to ribosomal protein S12, therefore, it was designated HmaS12. Slightly different results compared to published sequence data were obtained for the protein HS12 and HmaS19. The putative 'ribosomal' protein HSH could not be localized in the two-dimensional pattern of the total 30S ribosomal subunit proteins of H. marismortui. Therefore, it seems to be unlikely that this protein is a real constituent of the H. marismortui ribosome.
The clustered organization of most imprinted genes in mammals suggests coordinated genetic and epigenetic control mechanisms. Comparisons between human and mouse will help in elucidating these mechanisms by identifying structural and functional similarities. Previously we reported on such a comparison in the central part of the mouse imprinting cluster on distal chromosome 7 with the homologous Beckwith-Wiedemann syndrome (BWS) gene cluster on human chromosome 11p15.5. Here we focus on the adjacent sequences of 0.5 Mb including the KCNQ1/Kcnq1 and CDKN1C/Cdkn1c genes, which are implicated in BWS, and on one of the proposed boundary regions of the imprinting cluster. As in the previously analysed central region, this part of the cluster exhibits a highly conserved arrangement and structure of genes. The most striking similarity is found in the 3' part of the KCNQ1/Kcnq1 genes in large stretches of mostly non-coding sequences. The conserved region includes the recently identified KCNQ1OT1/Kcnq1ot1 antisense transcripts, flanked by a strikingly conserved cluster of LINE/Line elements and a CpG island which we show to carry a maternal germline methylation imprint. This region is likely to be the proposed second imprinting centre (IC2) in the BWS cluster. We also identified several novel genes inside and outside the previously proposed boundaries of the imprinting cluster. One of the genes outside the cluster, Obph1, is imprinted in mouse placenta indicating that at least in extra-embryonic tissues the imprinting cluster extends into a larger domain.
Preventing the formation of insoluble polyglutamine containing protein aggregates in neurons may represent an attractive therapeutic strategy to ameliorate Huntington's disease (HD). Therefore, the ability to screen for small molecules that suppress the self-assembly of huntingtin would have potential clinical and significant research applications. We have developed an automated filter retardation assay for the rapid identification of chemical compounds that prevent HD exon 1 protein aggregation in vitro . Using this method, a total of 25 benzothiazole derivatives that inhibit huntingtin fibrillogenesis in a dose-dependent manner were discovered from a library of ≈184,000 small molecules. The results obtained by the filter assay were confirmed by immunoblotting, electron microscopy, and mass spectrometry. Furthermore, cell culture studies revealed that 2-amino-4,7-dimethyl-benzothiazol-6-ol, a chemical compound similar to riluzole, significantly inhibits HD exon 1 aggregation in vivo . These findings may provide the basis for a new therapeutic approach to prevent the accumulation of insoluble protein aggregates in Huntington's disease and related glutamine repeat disorders.
Amyloid formation is widely accepted to play a crucial role in the pathogenesis of many neurodegenerative disorders including Alzheimer's disease (AD) and polyglutamine disorders such as Huntington's disease. The disease-specific amyloid-forming proteins share no sequence homologies and have different functions. However, recent studies demonstrated strong similarities in the process of protein aggregation despite the lack of identical sequences. For example, oligomeric intermediates made up of amyloid-beta, alpha-synuclein, polyglutamine protein, or other disease-associated amyloidogenic proteins are recognized by the same antibody and exert toxicity irrespective of the identity of the monomeric protein.
Activation of caspase-6 in the striatum of both presymptomatic and affected persons with Huntington's disease (HD) is an early event in the disease pathogenesis. However, little is known about the role of caspase-6 outside the central nervous system (CNS) and whether caspase activation might play a role in the peripheral phenotypes, such as muscle wasting observed in HD. We assessed skeletal muscle tissue from HD patients and well-characterized mouse models of HD. Cleavage of the caspase-6 specific substrate lamin A is significantly increased in skeletal muscle obtained from HD patients as well as in muscle tissues from two different HD mouse models. p53, a transcriptional activator of caspase-6, is upregulated in neuronal cells and tissues expressing mutant huntingtin. Activation of p53 leads to a dramatic increase in levels of caspase-6 mRNA, caspase-6 activity and cleavage of lamin A. Using mouse embryonic fibroblasts (MEFs) from YAC128 mice, we show that this increase in caspase-6 activity can be mitigated by pifithrin-α (pifα), an inhibitor of p53 transcriptional activity, but not through the inhibition of p53′s mitochondrial pro-apoptotic function. Remarkably, the p53-mediated increase in caspase-6 expression and activation is exacerbated in cells and tissues of both neuronal and peripheral origin expressing mutant huntingtin (Htt). These findings suggest that the presence of the mutant Htt protein enhances p53 activity and lowers the apoptotic threshold, which activates caspase-6. Furthermore, these results suggest that this pathway is activated both within and outside the CNS in HD and may contribute to both loss of CNS neurons and muscle atrophy.
Genomic imprinting is an epigenetically controlled form of gene regulation leading to the preferential expression of one parental gene copy. To date, approximately 40 imprinted genes have been described that are exclusively or predominantly expressed from either the paternal or the maternal allele (www.mgu.har.mrc.ac.uk/imprinting/implink.html). Changes in the imprinted expression of such genes result in developmental abnormalities; in the human they are associated with several diseases and various types of cancer (1-3).
Imprinted genes provide an excellent experimental system to examine the epigenetic control of gene expression. This is, first, because an active and inactive allele of the same gene are present in the same cell, so that the differentiation between expressed and nonexpressed status involves cis -acting genetic elements, and, second, because there must exist a long-lasting memory of the origin of alleles (from sperm or egg) that is sustained through numerous rounds of replication. Here we examine the epigenetic features of the imprinted insulin-like growth factor 2 (Igf2) gene and postulate that some of the methylation and chromatin features in this gene are influenced or controlled by methylation and chromatin modifier genes. In the second part of the chapter, we describe a genetic system for the identification and potential isolation of such modifiers of methylation and chromatin. In mouse and human, the Igf2 gene is almost exclusively expressed from the paternal chromosome (DeChiara et al. 1991; Giannoukakis et al. 1993; Ohlsson et al. 1993). For a number of reasons, this particular gene is important for the study of mechanisms and consequences of imprinting. First, Igf2 has a very strong effect on fetal growth, one of the key phenotypes controlled by imprinted genes. Second, there are at least two other (maternally expressed) imprinted genes, Igf2r and H19, that interact with the Igf2 pathway (Wang et al. 1994; Leighton et al. 1995a). Third, the Igf2 region of imprinted genes is implicated in a number of human diseases, most notably the fetal overgrowth and...
Huntington disease (HD) is caused by the expression of mutant huntingtin (mHTT) bearing a polyglutamine expansion. In HD, mHTT accumulation is accompanied by a dysfunction in basal autophagy, which manifests as specific defects in cargo loading during selective autophagy. Here we show that the expression of mHTT resistant to proteolysis at the caspase cleavage site D586 (C6R mHTT) increases autophagy, which may be due to its increased binding to the autophagy adapter p62. This is accompanied by faster degradation of C6R mHTT in vitro and a lack of mHTT accumulation the C6R mouse model with age. These findings may explain the previously observed neuroprotective properties of C6R mHTT. As the C6R mutation cannot be easily translated into a therapeutic approach, we show that a scheduled feeding paradigm is sufficient to lower mHTT levels in YAC128 mice expressing cleavable mHTT. This is consistent with a previous model, where the presence of cleavable mHTT impairs basal autophagy, while fasting-induced autophagy remains functional. In HD, mHTT clearance and autophagy may become increasingly impaired as a function of age and disease stage, because of gradually increased activity of mHTT-processing enzymes. Our findings imply that mHTT clearance could be enhanced by a regulated dietary schedule that promotes autophagy.
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