The Role of G Protein Activation in the Toxicity of Amyloidogenic Aβ-(1–40), Aβ-(25–35), and Bovine Calcitonin
2001; Elsevier BV; Volume: 276; Issue: 4 Linguagem: Inglês
10.1074/jbc.m005800200
ISSN1083-351X
AutoresDawn L. Rymer, Theresa A. Good,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoMore than 16 different proteins have been identified as amyloid in clinical diseases; among these, β-amyloid (Aβ) of Alzheimer's disease is the best characterized. In the present study, we performed experiments with Aβ and calcitonin, another amyloid-forming peptide, to examine the role of G protein activation in amyloid toxicity. We demonstrated that the peptides, when prepared under conditions that promoted β-sheet and amyloid fibril (or protofibril) formation, increased high affinity GTPase activity, but the nonamyloidogenic peptides had no discernible effects on GTP hydrolysis. These increases in GTPase activity were correlated to toxicity. In addition, G protein inhibitors significantly reduced the toxic effects of the amyloidogenic Aβ and calcitonin peptides. Our results further indicated that the amyloidogenic peptides significantly increased GTPase activity of purified Gαo and Gαi subunits and that the effect was not receptor-mediated. Collectively, these results imply that the amyloidogenic structure, regardless of the actual peptide or protein sequence, may be sufficient to cause toxicity and that toxicity is mediated, at least partially, through G protein activation. Our abilities to manipulate G protein activity may lead to novel treatments for Alzheimer's disease and the other amyloidoses. More than 16 different proteins have been identified as amyloid in clinical diseases; among these, β-amyloid (Aβ) of Alzheimer's disease is the best characterized. In the present study, we performed experiments with Aβ and calcitonin, another amyloid-forming peptide, to examine the role of G protein activation in amyloid toxicity. We demonstrated that the peptides, when prepared under conditions that promoted β-sheet and amyloid fibril (or protofibril) formation, increased high affinity GTPase activity, but the nonamyloidogenic peptides had no discernible effects on GTP hydrolysis. These increases in GTPase activity were correlated to toxicity. In addition, G protein inhibitors significantly reduced the toxic effects of the amyloidogenic Aβ and calcitonin peptides. Our results further indicated that the amyloidogenic peptides significantly increased GTPase activity of purified Gαo and Gαi subunits and that the effect was not receptor-mediated. Collectively, these results imply that the amyloidogenic structure, regardless of the actual peptide or protein sequence, may be sufficient to cause toxicity and that toxicity is mediated, at least partially, through G protein activation. Our abilities to manipulate G protein activity may lead to novel treatments for Alzheimer's disease and the other amyloidoses. β-amyloid phosphate-buffered saline triethanolamine 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide pertussis toxin guanosine 5′-O-(2-thiodiphosphate) 1,2-dipalmytoyl-sn-glycero-3-phosphocholine adenosine 5′-(β,γ-imino)triphosphate The amyloidoses are complex, multiform disorders characterized by the polymerization and aggregation of normally innocuous and soluble proteins or peptides into extracellular insoluble fibrils. More than 16 biochemically unique proteins, including transthyretin, α-synuclein, calcitonin, β2-macroglobulin, gelsolin, amylin, and β-amyloid, have been isolated as the fibrillar components of disease-associated amyloid deposits (1Kelly J.W. Curr. Opin. Struct. Biol. 1996; 6: 11-17Crossref PubMed Scopus (571) Google Scholar, 2Kelly J.W. Curr. Opin. Struct. Biol. 1998; 8: 101-106Crossref PubMed Scopus (951) Google Scholar, 3Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3342-3344Crossref PubMed Scopus (513) Google Scholar, 4Sipe J.D. Annu. Rev. Biochem. 1992; 61: 947-975Crossref PubMed Scopus (408) Google Scholar, 5Sipe J.D. Crit. Rev. Clin. Lab. Sci. 1994; 31: 325-354Crossref PubMed Scopus (191) Google Scholar). These proteins share no conserved primary structural motives or other structural homologies, but their fibrils all possess some common structural features (3Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3342-3344Crossref PubMed Scopus (513) Google Scholar, 6Kelly J.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 930-932Crossref PubMed Scopus (118) Google Scholar,7Taubes G. Science. 1996; 271: 1493-1495Crossref PubMed Scopus (131) Google Scholar). All amyloid fibrils contain β-sheet structures in which the polypeptide chains are orthogonally aligned in the fibril directions (8Glenner G.G. Terry W.D. Annu. Rev. Med. 1974; 25: 131-135Crossref PubMed Scopus (18) Google Scholar, 9Glenner G.G. N. Engl. J. Med. 1980; 302: 1283-1292Crossref PubMed Scopus (1307) Google Scholar, 10Glenner G.G. N. Engl. J. Med. 1980; 302: 1333-1343Crossref PubMed Scopus (611) Google Scholar). With Congo Red staining, amyloids show a green birefringence under polarized light, and under electron microscopy, their morphology consists of bundles of nonbranching, long filaments about 5–12 nm wide (4Sipe J.D. Annu. Rev. Biochem. 1992; 61: 947-975Crossref PubMed Scopus (408) Google Scholar, 5Sipe J.D. Crit. Rev. Clin. Lab. Sci. 1994; 31: 325-354Crossref PubMed Scopus (191) Google Scholar, 11Castano E.M. Frangione B. Curr. Opin. Neurol. 1995; 8: 279-285Crossref PubMed Scopus (17) Google Scholar). The most characterized amyloid-forming peptide is β-amyloid (Aβ)1 of Alzheimer's disease. The toxicity of Aβ has been directly linked to structure and amyloid content. In an aggregated state (containing fibrils, protofibrils, and low molecular weight intermediates), Aβ has been consistently shown to be toxic to neurons in culture (12Walsh D.M. Hartley D.M. Kusumoto Y. Fezoui Y. Condron M.M. Lomakin A. Benedek G.B. Selkoe D.J. Teplow D.B. J. Biol. Chem. 1999; 274: 25945-25952Abstract Full Text Full Text PDF PubMed Scopus (984) Google Scholar, 13Howlett D.R. Jennings K.H. Lee D.C. Clark M.S. Brown F. Wetzel R. Wood S.J. Camilleri P. Roberts G.W. Neurodegeneration. 1995; 4: 23-32Crossref PubMed Scopus (188) Google Scholar, 14Ward R.V. Jennings K.H. Jepras R. Neville W. Owen D.E. Hawkins J. Christie G. Davis J.B. George A. Karran E.H. Howlett D.R. Biochem. J. 2000; 348: 137-144Crossref PubMed Scopus (151) Google Scholar, 15Seilheimer B. Bohrmann B. Bondolfi L. Muller F. Stuber D. Dobeli H. J. Struct. Biol. 1997; 119: 59-71Crossref PubMed Scopus (202) Google Scholar, 16Yankner B.A. Duffy L.K. Kirschner D.A. Science. 1990; 250: 279-282Crossref PubMed Scopus (1910) Google Scholar, 17Busciglio J. Yeh J. Yankner B.A. J. Neurochem. 1993; 61: 1565-1568Crossref PubMed Scopus (84) Google Scholar, 18Koh J.Y. Yang L.L. Cotman C.W. Brain Res. 1990; 533: 315-320Crossref PubMed Scopus (610) Google Scholar). Although there is some disagreement as to the exact structure of the aggregated species associated with toxicity, whether it be a protofibril (14Ward R.V. Jennings K.H. Jepras R. Neville W. Owen D.E. Hawkins J. Christie G. Davis J.B. George A. Karran E.H. Howlett D.R. Biochem. J. 2000; 348: 137-144Crossref PubMed Scopus (151) Google Scholar, 19Hartley D.M. Walsh D.M. Ye C.P. Diehl T. Vasquez S. Vassilev P.M. Teplow D.B. Selkoe D.J. J. Neurosci. 1999; 19: 8876-8884Crossref PubMed Google Scholar), a diffusible, nonfibrillar ligand (20Lambert M.P. Barlow A.K. Chromy B.A. Edwards C. Freed R. Liosatos M. Morgan T.E. Rozovsky I. Trommer B. Viola K.L. Wals P. Zhang C. Finch C.E. Krafft G.A. Klein W.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6448-6453Crossref PubMed Scopus (3128) Google Scholar), or some other low molecular weight intermediate (19Hartley D.M. Walsh D.M. Ye C.P. Diehl T. Vasquez S. Vassilev P.M. Teplow D.B. Selkoe D.J. J. Neurosci. 1999; 19: 8876-8884Crossref PubMed Google Scholar), toxicity is associated with peptide structures that are part of the aggregation pathway associated with amyloid formation. In addition, Aβ neurotoxicity has been shown to be attenuated by Congo Red and rifampicin, which bind to and selectively inhibit the formation of Aβ amyloid fibrils (21Burgevin M.C. Passat M. Daniel N. Capet M. Doble A. Neuroreport. 1994; 5: 2429-2432Crossref PubMed Scopus (50) Google Scholar, 22Lorenzo A. Yankner B.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12243-12247Crossref PubMed Scopus (1298) Google Scholar, 23Sadler I.I. Smith D.W. Shearman M.S. Ragan C.I. Tailor V.J. Pollack S.J. Neuroreport. 1995; 7: 49-53Crossref PubMed Scopus (28) Google Scholar, 24Tomiyama T. Asano S. Suwa Y. Morita T. Kataoka K. Mori H. Endo N. Biochem. Biophys. Res. Commun. 1994; 204: 76-83Crossref PubMed Scopus (160) Google Scholar). Clearly, all of these observations imply a causal link between Aβ fibril formation and neurodegeneration. Various research groups have hypothesized potential molecular mechanisms of β-amyloid toxicity, but there is no consensus. Cellular responses to Aβ that have been postulated to result in toxicity encompass destabilization of calcium homeostasis, membrane depolarization, increased vulnerability to excitotoxins, increased membrane permeability due to free radical generation, blockage or functional loss of potassium channels, and direct disruption of membrane integrity (17Busciglio J. Yeh J. Yankner B.A. J. Neurochem. 1993; 61: 1565-1568Crossref PubMed Scopus (84) Google Scholar, 18Koh J.Y. Yang L.L. Cotman C.W. Brain Res. 1990; 533: 315-320Crossref PubMed Scopus (610) Google Scholar, 25–36). The preceding plethora of observed biochemical responses to Aβ suggests that perhaps a more common, fundamental pathway is initially being activated and that this pathway subsequently diverges to produce many unique intracellular responses. Analogous to Aβ, calcitonin is another model amyloid peptide associated with medullary carcinoma of the thyroid (37Berger G. Berger N. Guillaud M.H. Trouillas J. Vauzelle J.L. Virchows Arch. A Pathol. Anat. Histopathol. 1988; 412: 543-551Crossref PubMed Scopus (23) Google Scholar, 38Butler M. Khan S. Arch. Pathol. Lab. Med. 1986; 110: 647-649PubMed Google Scholar, 39Byard R.W. Thorner P.S. Chan H.S. Griffiths A.M. Cutz E. Pediatr. Pathol. 1990; 10: 581-592Crossref PubMed Scopus (17) Google Scholar, 40Dammrich J. Ormanns W. Schaffer R. Histochemistry. 1984; 81: 369-372Crossref PubMed Scopus (8) Google Scholar, 41DeLellis R.A. Nunnemacher G. Bitman W.R. Gagel R.F. Tashjian Jr., A.H. Blount M. Wolfe H.J. Lab. Invest. 1979; 40: 140-154PubMed Google Scholar, 42Silver M.M. Hearn S.A. Lines L.D. Troster M. J. Histochem. Cytochem. 1988; 36: 1031-1036Crossref PubMed Scopus (27) Google Scholar, 43Sletten K. Westermark P. Natvig J.B. J. Exp. Med. 1976; 143: 993-998Crossref PubMed Scopus (187) Google Scholar). The fibrils of human calcitonin have also been shown to be neurotoxic (44Schubert D. Behl C. Lesley R. Brack A. Dargusch R. Sagara Y. Kimura H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1989-1993Crossref PubMed Scopus (369) Google Scholar, 45Liu Y. Schubert D. J. Neurochem. 1997; 69: 2285-2293Crossref PubMed Scopus (175) Google Scholar, 46Koo E.H. Lansbury Jr., P.T. Kelly J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9989-9990Crossref PubMed Scopus (599) Google Scholar), suggesting that the amyloidoses may possess a shared mechanism of toxicity related to their secondary and macromolecular structures. To explore if a common, fundamental mechanism of toxicity exists in the amyloidoses, we examined the structure-function relationships of several synthetic Aβ sequences, Aβ-(1–40), Aβ-(25–35), Aβ-(1–16), and bovine calcitonin. We were able to manipulate the secondary and macromolecular structures of these peptides to produce stable amyloid and nonamyloid structures. With these model systems, we demonstrated that the peptides in an amyloid state (with high β-sheet content and the ability to bind Congo Red) altered G protein activity associated with both cell membrane extracts and purified Gα subunits. We showed that the abilities of the peptides to induce GTPase activation were correlated with their toxicities, and the neurotoxicities of the peptides were attenuated by specific and nonspecific GTPase inhibitors. In addition, we demonstrated that significant GTPase activities were still induced even when the cell surface receptors were removed with a nonspecific protease. These results suggest that G protein activation, possibly induced via a protein-membrane interaction, plays an important role in the toxicity of Aβ and other amyloid-forming proteins. Aβ-(1–40), Aβ-(25–35), and Aβ-(1–16) were purchased from BIOSOURCE International (Camarillo, CA), and bovine calcitonin was obtained from Sigma. ATP and GTP were purchased from Aldrich, and [γ-32P]GTP was from ICN Biochemicals (Irvine, CA). Suramin and Pronase were acquired from Calbiochem and Roche Molecular Biochemicals, respectively. Cell culture reagents were purchased from Life Technologies, Inc. Purified 1,2-dipalmytoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol were obtained from Avanti Polar Lipids (Alabaster, AL). The XK16/70 column and Superfine Sephadex G-50 for size exclusion were acquired from Amersham Pharmacia Biotech. Purified Go and Gi α subunits and epinephrine were purchased from Calbiochem. All other chemicals, unless otherwise specified, were obtained from Sigma. The Aβ peptides were prepared analogously to established methods in the toxicity and structural literature for forming β-sheet structures and fibril formations (47Gorevic P.D. Castano E.M. Sarma R. Frangione B. Biochem. Biophys. Res. Commun. 1987; 147: 854-862Crossref PubMed Scopus (138) Google Scholar, 48Shen C.L. Fitzgerald M.C. Murphy R.M. Biophys. J. 1994; 67: 1238-1246Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 49Shen C.L. Murphy R.M. Biophys. J. 1995; 69: 640-651Abstract Full Text PDF PubMed Scopus (273) Google Scholar, 50Whitson J.S. Mims M.P. Strittmatter W.J. Yamaki T. Morrisett J.D. Appel S.H. Biochem. Biophys. Res. Commun. 1994; 199: 163-170Crossref PubMed Scopus (65) Google Scholar). Stock solutions of 10 mg/ml were prepared by dissolving the Aβ peptides in 0.1% (v/v) trifluoroacetic acid in water. After incubating for 1 h at 25 °C, the peptide stock solutions were diluted to concentrations of 0.5 mg/ml in sterile phosphate-buffered saline (PBS) (0.01 m NaH2PO4, 0.15m NaCl, pH 7.4) with antibiotics. These solutions were rotated on a model RD4524 rotator (Glas-col, Terre Haute, IN) at 60 rpm at 25 °C for 24 h. The peptides were then diluted to final concentrations of 20 μm in sterile medium and rotated for an additional 24 h prior to being added to the culture wells or plates for the toxicity and GTP studies. Bovine calcitonin was directly dissolved in various solvents and buffers at concentrations of 40 and 80 μm. CD measurements of the solutions were recorded 2–24 h later on a model 62DS spectrometer (Aviv Instruments, Lakewood, NJ) at 25 °C using a bandwidth of 1.0 nm, a step interval of 0.5 nm, and an averaging time of 2 s. A 0.01-cm quartz cell was used for the far-UV (190–250 nm) measurements. The instrument was calibrated usingd(+)-10-camphorsulfonic acid. Three scans each of duplicate samples were measured and averaged. Control buffer and solvent scans were run in duplicate, averaged, and then subtracted from the sample spectra. Spectra were analyzed using the secondary structural parameters reported by Chang (51Chang C.T. Wu C.S. Yang J.T. Anal. Biochem. 1978; 91: 13-31Crossref PubMed Scopus (1025) Google Scholar) to ascertain the sample percentages of α-helix, β-sheet, β-turn, and random-coil. To assess the presence of amyloid fibrils in the calcitonin solutions, Congo Red binding studies were performed. Congo Red dye was dissolved in PBS to a final concentration of 112 μm. Congo Red absorbances of the calcitonin solutions and free dye controls were determined by adding Congo Red to a final concentration of 12 μm and acquiring spectral measurements from 300 to 900 nm at 25 °C on a model 420 UV-visible spectrophotometer (Spectral Instruments, Tucson, AZ) (52Klunk W.E. Pettegrew J.W. Abraham D.J. J. Histochem. Cytochem. 1989; 37: 1293-1297Crossref PubMed Scopus (113) Google Scholar, 53Klunk W.E. Pettegrew J.W. Abraham D.J. J. Histochem. Cytochem. 1989; 37: 1273-1281Crossref PubMed Scopus (568) Google Scholar). Both the calcitonin solutions and the control solutions were allowed to interact with Congo Red for 1 h prior to recording their spectra. Congo Red difference spectra were calculated by subtracting the free dye absorbance from the calcitonin-dye absorbances. Finally, bovine calcitonin was prepared in the following manner for the toxicity and GTP studies. Calcitonin was dissolved at a concentration of 1.5 mg/ml in either deionized water or a solution of 5 mm CaCl2 and 1 mm MgCl2in water. These solutions were rotated at 60 rpm at 25 °C for 24 h. Then the calcitonin solutions were diluted to 40 and 80 μm with sterile medium and rotated for an additional 24 h prior to being added to the culture wells or plates for the toxicity and GTP studies. Human neuroblastoma SH-SY5Y cells (a gift of Dr. Evelyn Tiffany-Castiglioni, College of Veterinary Medicine, Texas A & M University, College Station, TX) were cultured in a humidified 5% (v/v) CO2/air environment at 37 °C in minimum Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 3 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin B (fungizone). Likewise, rat pheochromocytoma PC12 cells (ATCC, Manassas, VA) were cultured in RPMI medium supplemented with 10% (v/v) horse serum, 5% (v/v) fetal bovine serum, 3 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml fungizone in a 5% (v/v) CO2/air environment at 37 °C. For the GTP studies, cells were plated at densities ranging from 2.5 to 4 million cells in 35-mm tissue culture dishes. For the viability assays, cells were plated at a density of 1 × 105cells/well in 96-well plates. During both the GTP and viability studies, the peptides were added to the cells 24 h after plating. After incubation with the peptides or controls for 30 min at 37 °C, the PC12 or SH-SY5Y membranes were isolated using the widely accepted method of Seifert and Schultz (54Seifert R. Schultz G. Biochem. Biophys. Res. Commun. 1987; 146: 1296-1302Crossref PubMed Scopus (46) Google Scholar). Cells were harvested with a cell scraper and collected by centrifugation (1600 × g, 4 °C, 20 min). Subsequently, they were washed with a buffer consisting of 10 mm triethanolamine (TEA) and 140 mm NaCl (pH 7.4) and disrupted by nitrogen cavitation in a 50 mmKH2PO4 buffer with 100 mm NaCl, 3 mm EDTA, and 15 mm β-mercaptoethanol (pH 7.0). The nuclear portion of the cells was removed by a short centrifugation (1000 × g, 4 °C, 2 min), and membrane sedimentation was attained with a long centrifugation (15,000 × g, 4 °C, 60 min). The resulting membrane pellet was suspended in a 10 mm TEA/HCl buffer (pH 7.4), and the total membrane protein content was measured with the BCA assay (55Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18647) Google Scholar). The GTPase activities of the PC12 and SH-SY5Y membranes were assessed similarly to procedures described previously (56Cassel D. Selinger Z. Biochim. Biophys. Acta. 1976; 452: 538-551Crossref PubMed Scopus (404) Google Scholar, 57Aktories K. Schultz G. Jakobs K.H. Naunyn-Schmiedeberg's Arch. Pharmacol. 1983; 324: 196-200Crossref PubMed Scopus (30) Google Scholar, 58Brouillet E. Trembleau A. Galanaud D. Volovitch M. Bouillot C. Valenza C. Prochiantz A. Allinquant B. J. Neurosci. 1999; 19: 1717-1727Crossref PubMed Google Scholar, 59Klinker J.F. Lichtenberg-Kraag B. Damm H. Rommelspacher H. Neurosci. Lett. 1996; 213: 25-28Crossref PubMed Scopus (15) Google Scholar, 60Offermanns S. Schultz G. Rosenthal W. Eur. J. Biochem. 1989; 180: 283-287Crossref PubMed Scopus (18) Google Scholar). Reaction mixtures of 100 μl consisted of 0.4 μm [γ-32P]GTP (0.5 μCi/tube), 0.5 mm MgCl2, 0.1 mm EGTA, 0.1 mm ATP, 1 mm AMP-PNP, 5 mm creatine phosphate, 40 μg of creatine kinase, 1 mm dithiothreitol, and 0.2% (w/v) bovine serum albumin in 50 mm TEA/HCl (pH 7.4). Following a 5-min preincubation period at 25 °C, the reaction was initiated by the addition of 5–8 μg of membrane protein. After 15 min at 25 °C, the reaction was stopped by the addition of 800 μl of a 20 mm KH2PO4 buffer (4 °C, pH 7.0) containing 5% (w/v) activated charcoal. The released32Pi was separated from the nucleotide-bound phosphate by centrifugation (15000 × g, 4 °C, 20 min), and 100 μl of the supernatant was counted on a Topcount Microplate Scintillation Counter (Packard Instrument Co.). Low affinity or nonspecific GTPase activity was measured by adding excess unlabeled GTP (50 μm) to the aforementioned reaction mixture and conducting the reaction as described. Specific high affinity GTPase activity was calculated as the difference between the total GTPase activity in the absence of unlabeled GTP and the low affinity GTPase activity. Pronase studies were conducted analogously to established procedures (61Lazari M.F. Porto C.S. Freymuller E. Abreu L.C. Picarelli Z.P. Biochem. Pharmacol. 1997; 54: 399-408Crossref PubMed Scopus (14) Google Scholar, 62Dorland R.B. Middlebrook J.L. Leppla S.H. J. Biol. Chem. 1979; 254: 11337-11342Abstract Full Text PDF PubMed Google Scholar, 63Trop M. Birk Y. Biochem. J. 1970; 116: 19-25Crossref PubMed Scopus (71) Google Scholar). Pronase at a concentration of 3 mg/ml in serum-free RPMI was incubated with the plated PC12 cells for 1 h at 4 °C. The cells were harvested with a cell scraper and collected by centrifugation (1600 ×g, 4 °C, 20 min). Then the cells were thoroughly washed with PBS and centrifuged again (1600 × g, 4 °C, 20 min) prior to the addition of the peptides or controls for the GTPase assays. For the epinephrine control, 200 μm epinephrine in serum-free RPMI was incubated with these Pronase-treated PC12 cells for 30 min prior to the membrane isolation step. SH-SY5Y and PC12 cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. MTT is reduced by viable cells to form blue formazan crystals, and inhibition of this reaction is indicative of cellular redox alterations that could result in toxicity (64Pollack S.J. Sadler I.I. Hawtin S.R. Tailor V.J. Shearman M.S. Neurosci. Lett. 1995; 184: 113-116Crossref PubMed Scopus (102) Google Scholar). The peptides were incubated with the SH-SY5Y and PC12 cells for 24 h, after which time MTT reduction was assessed. MTT was added to the culture medium to yield a final concentration of 0.5 mg/ml. The cells were allowed to incubate with the MTT for 4 h in a CO2incubator after which time 100 μl of a 5:2:3N,N-dimethylformamide/SDS/water solution (pH 4.7) was added to dissolve the formed formazan crystals. After 18 h of incubation in a humidified CO2 incubator, the results were read using an Emax Microplate reader at 585 nm (Molecular Devices, Sunnyvale, CA). Viability is reported relative to control cells unexposed to the peptides. Pertussis toxin (PT) (100 ng/ml), GDPβS (600 μm), and suramin (20 μm) were incubated with the PC12 and SH-SY5Y cells for 24, 3, and 3 h, respectively, at 37 °C prior to the peptide additions for the GTPase or toxicity assays. The peptide solutions for these assays also contained the same inhibitors at the same concentrations. Control cells were treated identically except for the presence of peptide. Vesicles consisted of 82% (w/w) DPPC and 18% (w/w) cholesterol, and they were prepared by mixing the DPPC and cholesterol in chloroform and evaporating off the solvent under nitrogen at 50 °C in a 421–4000 Micro Rotary Evaporator (Labconco, Kansas City, MO). 20 mm NaHepes (pH 8.0) containing 0.4% (w/v) deoxycholate and 0.04% (w/v) cholate was then added to suspend the lipid film, producing a final lipid concentration of 1 mg/ml. The resulting DPPC/cholesterol suspension was sonicated for 10 min. Subsequently, 1.2 volumes of these DPPC/cholesterol vesicles were combined with 0.6 volume of Gαo or Gαi in a 10 mm NaHepes buffer (pH 8.0) containing 1 mmEDTA, 0.1 mm dithiothreitol, and 0.1% (v/v) Genapol. This mixture was gel-filtered using an ÄKTA Explorer (Amersham Pharmacia Biotech) with Sephadex G-50 in a XK16/70 column at a flow rate of 0.5 ml/min according to procedure of Pedersen and Ross (65Pedersen S.E. Ross E.M. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7228-7232Crossref PubMed Scopus (60) Google Scholar). The elution buffer consisted of 20 mm NaHepes buffer (pH 8.0), 1 mm EDTA, 1 mm dithiothreitol, 0.1m NaCl, and 2 mm MgCl2. GTPase activity was assayed by incubating the Gαo or Gαi vesicles (5 μl; 75 fmol of Gαo or Gαi) at 30 °C in a total volume of 100 μl containing 50 mm NaHepes (pH 8.0), 1 mm EDTA, 1 mm dithiothreitol, 0.1 mNaCl, 2 mm MgCl2, 0.1 mm adenosine AMP-PNP, 0.1 mm ascorbic acid, and 0.1 μm[γ-32P]GTP. After the specified time, the reaction was stopped by the addition of 250 μl of a 50 mmNaH2PO4 buffer (4 °C, pH 7.0) with 5% (w/v) activated charcoal and rapid chilling. The mixture was centrifuged (1200 × g, 4 °C, 10 min), and the [32P]Pi in the supernatant (200 μl) was counted on a Topcount Microplate Scintillation Counter (Packard Instrument Co.). The significance of results was determined using a Student's t test on nindependent measurements, where n is specified in the figure legend. Unless otherwise indicated, significance was taken asp < 0.05. The structures of the Aβ peptides under the employed solvation conditions have been well characterized and were not re-examined. Under these conditions, Aβ-(1–40) and Aβ-(25–35) have been shown to be amyloidogenic (containing fibrils and protofibrils) and to contain extensive β-sheet structures, whereas Aβ-(1–16) has been demonstrated to be nonamyloidogenic and predominantly random coil (47Gorevic P.D. Castano E.M. Sarma R. Frangione B. Biochem. Biophys. Res. Commun. 1987; 147: 854-862Crossref PubMed Scopus (138) Google Scholar, 48Shen C.L. Fitzgerald M.C. Murphy R.M. Biophys. J. 1994; 67: 1238-1246Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 49Shen C.L. Murphy R.M. Biophys. J. 1995; 69: 640-651Abstract Full Text PDF PubMed Scopus (273) Google Scholar, 50Whitson J.S. Mims M.P. Strittmatter W.J. Yamaki T. Morrisett J.D. Appel S.H. Biochem. Biophys. Res. Commun. 1994; 199: 163-170Crossref PubMed Scopus (65) Google Scholar). Because the solution structures of bovine calcitonin are not as well documented, we identified conditions that promoted the formation of β-sheet structure and amyloid using CD spectroscopy and Congo Red binding assays. As determined by CD (Fig.1 A), both 40 and 80 μm bovine calcitonin in water containing 5 mmCaCl2 and 1 mm MgCl2 adopted structures with ∼55 ± 10% β-sheet and only 15 ± 10% α-helix. Incubating the peptide in deionized water alone at these same concentrations (Fig. 1 A) produced structures devoid of β-sheet character with 95 ± 10% α-helical contents. As depicted by Congo Red difference spectra (Fig. 1 B), the 40 and 80 μm water solutions of bovine calcitonin with 5 mm CaCl2 and 1 mm MgCl2significantly bound and shifted the spectral properties of Congo Red, indicating the formation of amyloid. The zero difference spectrum of the peptide in deionized water indicated an absence of Congo Red binding and substantial amyloid fibril formation (spectrum not shown). By using Aβ-(1–40), Aβ-(25–35), and bovine calcitonin in water with 5 mm CaCl2 and 1 mm MgCl2 as models of peptides with amyloidogenic structures and Aβ-(1–16) and bovine calcitonin in deionized water as models of peptides without amyloidogenic structures, we examined the relationship between peptide structure and GTPase activity. We found that the rate of high affinity GTP hydrolysis in PC12 membranes increased by 31 ± 12% on average with exposure to the peptides containing extensive β-sheet and amyloid contents relative to the rate of hydrolysis of control cells unexposed to peptides (Fig. 2 A). In all cases, the increases in GTPase activity were significant relative to the control cells (p < 0.001). The rates of GTP hydrolysis were significantly greater for Aβ-(25–35) and 80 μm calcitonin (p < 0.001), the two peptides with the greatest amyloid content, than for Aβ-(1–40) and 40 μm calcitonin, suggesting that the extent of macromolecular structure influences the process. Similarly, Aβ-(1–16) and bovine calcitonin in deionized water, the peptides devoid of amyloid and β-sheet structures, did not significantly alter the GTPase activities of the PC12 cells relative to untreated controls (Fig. 2 A) (p > 0.2). To ensure that the observed phenomena were not isolated to PC12 cells, we examined the GTPase activities of SH-SY5Y membranes exposed to bovine calcitonin. We found similar trends to those observed with the PC12 cells (Fig. 2 B). The rate of GTP hydrolysis increased from 16.0 ± 0.5 pmol/mg/min for the control cells to 23.8 ± 0.6 and 18.7 ± 0.6 pmol/mg/min for the cells exposed to 80 and 40 μm bovine calcitonin in water with 5 mmCaCl2 and 1 mm MgCl2, respectively (p < 0.002). The nonamyloidogenic 80 μmcalcitonin in water did not significantly alter GTPase activity relative to the controls (16.0 ± 0.3 pmol/mg/min,p > 0.4). In parallel to the GTPase activity experiments, we examined the relationship between peptide structure and toxicity using our model peptides. As seen in Fig. 3,A and B, analogous to our GTPase results, we found that exposure to the peptides containing extensive β-sheet and amyloid contents resulted in significant PC12 (Fig. 3 A) and SH-SY5Y (Fig. 3 B) cell toxicity (p < 0.001). Conversely, Aβ-(1–16) and bovine calcitonin in deionized water, the peptides devoid of amyloid and β-sheet structures, did not significantly alter cell viability relative to untreated controls (p > 0.2). To identify the family or families of G proteins activated by the amyloid-forming peptides, we investigated the effects of GDPβS and suramin, two nonspecific G pr
Referência(s)