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ABSTRACT: The ΔE693 (Japanese) mutation of the β-amyloid precursor protein leads to production of ΔE22-Aβ peptides such as ΔE22-Aβ(1-39). Despite reports that these peptides do not form fibrils, here we show that, on the contrary, the peptide forms fibrils essentially instantaneously. The fibrils are typical amyloid fibrils in all respects except that they cause only low levels of thioflavin T (ThT) fluorescence, which, however, develops with no lag phase. The fibrils bind ThT, but with a lower affinity and a smaller number of binding sites than wild-type (WT) Aβ(1-40). Fluorescence depolarization confirms extremely rapid aggregation of ΔE22-Aβ(1-39). Size exclusion chromatography (SEC) indicates very low concentrations of soluble monomer and oligomer, but only in the presence of some organic solvent, e.g., 2% (v/v) DMSO. The critical concentration is approximately 1 order of magnitude lower for ΔE22-Aβ(1-39) than for WT Aβ(1-40). Several lines of evidence point to an altered structure for ΔE22-Aβ(1-39) compared to that of WT Aβ(1-40) fibrils. In addition to differences in ThT binding and fluorescence, PITHIRDS-CT solid-state nuclear magnetic resonance (NMR) measurements of ΔE22-Aβ(1-39) are not compatible with the parallel in-register β-sheet generally observed for WT Aβ(1-40) fibrils. X-ray fibril diffraction showed different D spacings: 4.7 and 10.4 Å for WT Aβ(1-40) and 4.7 and 9.6 Å for ΔE22-Aβ(1-39). Equimolar mixtures of ΔE22-Aβ(1-39) and WT Aβ(1-40) also produced fibrils extremely rapidly, and by the criteria of ThT fluorescence and electron microscopic appearance, they were the same as fibrils made from pure ΔE22-Aβ(1-39). X-ray diffraction of fibrils formed from 1:1 molar mixtures of ΔE22-Aβ(1-39) and WT Aβ(1-40) showed the same D spacings as fibrils of the pure mutant peptide, not the wild-type peptide. These findings are consistent with extremely rapid nucleation by ΔE22-Aβ(1-39), followed by fibril extension by WT Aβ(1-40), and "conversion" of the wild-type peptide to a structure similar to that of the mutant peptide, in a manner reminiscent of the prion conversion phenomenon.
Biochemistry 02/2011; 50(12):2026-39. · 3.42 Impact Factor
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Louise Scharf,
Nan-Sheng Li,
Andrew J Hawk,
Diana Garzón,
Tejia Zhang,
Lisa M Fox,
Allison R Kazen,
Sneha Shah,
Esmael J Haddadian,
Jenny E Gumperz,
Alan Saghatelian,
José D Faraldo-Gómez, Stephen C Meredith,
Joseph A Piccirilli,
Erin J Adams
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ABSTRACT: CD1 molecules function to present lipid-based antigens to T cells. Here we present the crystal structure of CD1c at 2.5 Å resolution, in complex with the pathogenic Mycobacterium tuberculosis antigen mannosyl-β1-phosphomycoketide (MPM). CD1c accommodated MPM's methylated alkyl chain exclusively in the A' pocket, aided by a unique exit portal underneath the α1 helix. Most striking was an open F' pocket architecture lacking the closed cavity structure of other CD1 molecules, reminiscent of peptide binding grooves of classical major histocompatibility complex molecules. This feature, combined with tryptophan-fluorescence quenching during loading of a dodecameric lipopeptide antigen, provides a compelling model by which both the lipid and peptide moieties of the lipopeptide are involved in CD1c presentation of lipopeptides.
Immunity 12/2010; 33(6):853-62. · 21.64 Impact Factor
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ABSTRACT: Polyglutamine expansion in the exon 1 domain of huntingtin leads to aggregation into beta-sheet-rich insoluble aggregates associated with Huntington's disease. We assessed eight polyglutamine peptides with different permutations of N-methylation of backbone and side chain amides as potential inhibitors of polyglutamine aggregation. Surprisingly, the most effective inhibitor, 5QMe(2) [Anth-K-Q-Q(Me(2))-Q-Q(Me(2))-Q-CONH(2), where Anth is N-methylanthranilic acid and Q(Me(2)) is side chain N-methyl Q], has only side chain methylations at alternate residues, highlighting the importance of side chain interactions in polyglutamine fibrillogenesis. Above a 1:1 stoichiometric ratio, 5QMe(2) can completely prevent fibrillation of a synthetic aggregating peptide, YAQ(12)A; it also shows significant inhibition at substoichiometric ratios. Surface plasmon resonance (SPR) measurements show a moderate K(d) with very fast k(on) and k(off) values. Sedimentation equilibrium analytical ultracentrifugation indicates that 5QMe(2) is predominantly or entirely monomeric at concentrations of <or=1 mM and that it forms a 1:1 stoichiometric complex with a fibril-forming target, YAQ(12)A. 5QMe(2) inhibits not only nucleation of YAQ(12)A but also fibril extension, as shown by the fact that it also inhibits seeded fibril growth where the nucleation steps are bypassed. 5QMe(2) acts on its targets only when they are in the PPII-like conformation, but not after they undergo a transition to beta-sheets. Thus, 5QMe(2) does not disassemble preformed YAQ(12)A; this contrasts with our previously described, backbone N-methylated inhibitors of beta-amyloid aggregation [Gordon, D. J., et al. (2001) Biochemistry 40, 8237-8245; Gordon, D. J., et al. (2002) J. Pept. Res. 60, 37-55]. The mode of action of 5QMe(2) is reminiscent of that of chaperones, because it binds and releases its targets very rapidly and maintains them in a nonaggregation-prone, monomeric state, in this case, the polyproline II (PPII)-like conformation, as shown by circular dichroism spectroscopy.
Biochemistry 08/2010; 49(33):7108-18. · 3.42 Impact Factor
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Nature Chemical Biology 01/2010; 6(1):7-8. · 14.69 Impact Factor
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ABSTRACT: Bacterial expression of full length beta-amyloid (Abeta) is problematic because of toxicity and poor solubility of the expressed protein, and a strong tendency of Met35 to become oxidized in inclusion bodies. We have developed a semisynthetic method in which Abeta1-29 is expressed in bacteria as part of a fusion protein with a C-terminal intein and Chitin-Binding Domain (CBD). There is also a single residue, N-terminal Met extension. The protein, Met-Abeta1-29-Intein-CBD, is well expressed and highly water-soluble. After binding of the expressed protein to Chitin beads, treatment with sodium 2-mercapto-ethane sulfonate (MESNA) yields Met-Abeta1-29-MESNA, with a C-terminal thioester suitable for native chemical ligation. Met-Abeta1-29-MESNA is first subjected to CNBr cleavage, which removes the N-terminal Met residue, but leaves the thioester intact. We synthesized NH2-A30C-Abeta30-40, which has an N-terminal Cys residue and is the partner for native chemical ligation with Met-Abeta1-29-MESNA. Native chemical ligation proceeds rapidly and efficiently (>90% yield) to give A30C-Abeta1-40. The final step is selective desulfurization using Raney-Ni, which also proceeds rapidly and efficiently (>90% yield) to give native sequence Abeta1-40. Overall, this system is highly efficient, and can yield approximately 8-10 mg of pure Abeta1-40 from one liter of bacterial culture medium. This procedure is adaptable for producing other Abeta peptides. We have also expressed an Abeta construct bearing a point mutation associated with one type of familial Alzheimer's Disease, the Iowa mutation, i.e., Met-D23N-Abeta1-29-Intein-CBD. Since expression of the intein-containing fusion protein is robust in minimal media as well as standard enriched media, this procedure also can be readily modified for incorporating 15N or 13C labels for NMR. Future work will also include extending this system to longer Abeta peptides, such as Abeta1-42.
Biopolymers 01/2010; 94(4):511-20. · 2.87 Impact Factor
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ABSTRACT: PolyQ peptides teeter between polyproline II (PPII) and beta-sheet conformations. In tandem polyQ-polyP peptides, the polyP segment tips the balance toward PPII, increasing the threshold number of Gln residues needed for fibrillation. To investigate the mechanism of cis-inhibition by flanking polyP segments on polyQ fibrillation, we examined short polyQ, polyP, and tandem polyQ-polyP peptides. These polyQ peptides have only three glutamines and cannot form beta-sheet fibrils. We demonstrate that polyQ-polyP peptides form small, soluble oligomers at high concentrations (as shown by size exclusion chromatography and diffusion coefficient measurements) with PPII structure (as shown by circular dichroism spectroscopy and (3)J(HN-C alpha) constants of Gln residues from constant time correlation spectroscopy NMR). Nuclear Overhauser effect spectroscopy and molecular modeling suggest that self-association of these peptides occurs as a result of both hydrophobic and steric effects. Pro side chains present three methylenes to solvent, favoring self-association of polyP through the hydrophobic effect. Gln side chains, with two methylene groups, can adopt a conformation similar to that of Pro side chains, also permitting self-association through the hydrophobic effect. Furthermore, steric clashes between Gln and Pro side chains to the C-terminal side of the polyQ segment favor adoption of the PPII-like structure in the polyQ segment. The conformational adaptability of the polyQ segment permits the cis-inhibitory effect of polyP segments on fibrillation by the polyQ segments in proteins such as huntingtin.
Biophysical Journal 10/2009; 97(8):2295-305. · 3.65 Impact Factor
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ABSTRACT: Asp23-to-Asn mutation within the coding sequence of beta-amyloid, called the Iowa mutation, is associated with early onset, familial Alzheimer's disease and cerebral amyloid angiopathy, in which patients develop neuritic plaques and massive vascular deposition predominantly of the mutant peptide. We examined the mutant peptide, D23N-Abeta40, by electron microscopy, X-ray diffraction, and solid-state NMR spectroscopy. D23N-Abeta40 forms fibrils considerably faster than the wild-type peptide (k = 3.77 x 10(-3) min(-1) and 1.07 x 10(-4) min(-1) for D23N-Abeta40 and the wild-type peptide WT-Abeta40, respectively) and without a lag phase. Electron microscopy shows that D23N-Abeta40 forms fibrils with multiple morphologies. X-ray fiber diffraction shows a cross-beta pattern, with a sharp reflection at 4.7 A and a broad reflection at 9.4 A, which is notably smaller than the value for WT-Abeta40 fibrils (10.4 A). Solid-state NMR measurements indicate molecular level polymorphism of the fibrils, with only a minority of D23N-Abeta40 fibrils containing the in-register, parallel beta-sheet structure commonly found in WT-Abeta40 fibrils and most other amyloid fibrils. Antiparallel beta-sheet structures in the majority of fibrils are indicated by measurements of intermolecular distances through (13)C-(13)C and (15)N-(13)C dipole-dipole couplings. An intriguing possibility exists that there is a relationship between the aberrant structure of D23N-Abeta40 fibrils and the unusual vasculotropic clinical picture in these patients.
Biochemistry 05/2009; 48(26):6072-84. · 3.42 Impact Factor
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ABSTRACT: Studies by solid-state nuclear magnetic resonance (NMR) of amyloid fibrils prepared in vitro from synthetic 40-residue beta-amyloid (Abeta(1-40)) peptides have shown that the molecular structure of Abeta(1-40) fibrils is not uniquely determined by amino acid sequence. Instead, the fibril structure depends on the precise details of growth conditions. The molecular structures of beta-amyloid fibrils that develop in Alzheimer's disease (AD) are therefore uncertain. We demonstrate through thioflavin T fluorescence and electron microscopy that fibrils extracted from brain tissue of deceased AD patients can be used to seed the growth of synthetic Abeta(1-40) fibrils, allowing preparation of fibrils with isotopic labeling and in sufficient quantities for solid-state NMR and other measurements. Because amyloid structures propagate themselves in seeded growth, as shown in previous studies, the molecular structures of brain-seeded synthetic Abeta(1-40) fibrils most likely reflect structures that are present in AD brain. Solid-state (13)C NMR spectra of fibril samples seeded with brain material from two AD patients were found to be nearly identical, indicating the same molecular structures. Spectra of an unseeded control sample indicate greater structural heterogeneity. (13)C chemical shifts and other NMR data indicate that the predominant molecular structure in brain-seeded fibrils differs from the structures of purely synthetic Abeta(1-40) fibrils that have been characterized in detail previously. These results demonstrate a new approach to detailed structural characterization of amyloid fibrils that develop in human tissue, and to investigations of possible correlations between fibril structure and the degree of cognitive impairment and neurodegeneration in AD.
Proceedings of the National Academy of Sciences 05/2009; 106(18):7443-8. · 9.68 Impact Factor
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ABSTRACT: In this paper, we describe two novel types of planar cyclic peptide templates for the facile addition of ligands that extend axially from the plane of the template ring. The first uses beta-amino acids of alternating D- and L-chirality, since the insertion of the additional methylene group in the peptide backbone was predicted and subsequently shown by NMR and molecular modeling, to reorient ligands attached to amino acid side chain axially with respect to the template ring. A second contains alternating D- and L-amino acids with an achiral Gly residue interposed between each chiral amino acid. The inserted Gly residues also tend to reorient side chains axially rather than radially, as was demonstrated by NMR and molecular modeling. The axial orientation of attached ligands is intended to foster or allow interactions among attached ligands in situations in which this is desired. Two such situations that we consider are (1) development of immunological reagents with avidity effects and (2) modeling of oligomers in fibril-forming peptides. Toward the first of these goals, we demonstrated that these templates are suitable for attaching macromolecules, by incorporating two types of protein, neutravidin and trypsinogen. Toward the second goal, we demonstrate the attachment of two different fibril-forming peptides to the template. The templates described herein thus have many of the desirable traits of such molecules, i.e., (1) multivalency for the attachment of multiple ligands, (2) suitable chemical functions for facile attachment of ligands, (3) versatility as to the number and spacing of ligand attachment sites, (4) sufficient rigidity so that the attached ligands can be similarly oriented with respect to the template, and (5) sufficient flexibility to allow even large ligands, such as proteins, to attach and interact.
Bioconjugate Chemistry 02/2009; 20(2):231-40. · 4.93 Impact Factor
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ABSTRACT: Humans have two major high density lipoprotein (HDL) sub-fractions, HDL(2) and HDL(3), whereas mice have a monodisperse HDL profile. Epidemiological evidence has suggested that HDL(2) is more atheroprotective; however, currently there is no direct experimental evidence to support this postulate. The amino acid sequence of apoA-I is a primary determinant of HDL subclass formation. The majority of the alpha-helical repeats in human apoA-I are proline-punctuated. A notable exception is the boundary between helices 7 and 8, which is located in the transitional segment between the stable N-terminal domain and the C-terminal hydrophobic domain. In this study we ask whether the substitution of a proline-containing sequence (PCS) separating other helices in human apoA-I for the non-proline-containing sequence (NPCS) between helices 7 and 8 (residues 184-190) influences HDL subclass association. The human apoA-I mutant with PCS2 replacing NPCS preferentially bound to HDL(2). In contrast, the mutant where PCS3 replaced NPCS preferentially associated with HDL(3). Thus, the specific amino acid sequence between helices 7 and 8 influences HDL subclass association. The wild-type and mutant proteins exhibited similar physicochemical properties except that the two mutants displayed greater lipid-associated stability versus wild-type human apoA-I. These results focus new attention on the influence of the boundary between helices 7 and 8 on the properties of apoA-I. The expression of these mutants in mice may result in the preferential generation of HDL(2) or HDL(3) and allow us to examine experimentally the anti-atherogenicity of the HDL subclasses.
Journal of Biological Chemistry 07/2008; 283(23):15779-88. · 4.77 Impact Factor
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ABSTRACT: beta-Rich self-assembly is a major structural class of polypeptides, but still little is known about its atomic structures and biophysical properties. Major impediments for structural and biophysical studies of peptide self-assemblies include their insolubility and heterogeneous composition. We have developed a model system, termed peptide self-assembly mimic (PSAM), based on the single-layer beta-sheet of Borrelia outer surface protein A. PSAM allows for the capture of a defined number of self-assembly-like peptide repeats within a water-soluble protein, making structural and energetic studies possible. In this work, we extend our PSAM approach to a highly hydrophobic peptide sequence. We show that a penta-Ile peptide (Ile(5)), which is insoluble and forms beta-rich self-assemblies in aqueous solution, can be captured within the PSAM scaffold in a form capable of self-assembly. The 1.1-A crystal structure revealed that the Ile(5) stretch forms a highly regular beta-strand within this flat beta-sheet. Self-assembly models built with multiple copies of the crystal structure of the Ile(5) peptide segment showed no steric conflict, indicating that this conformation represents an assembly-competent form. The PSAM retained high conformational stability, suggesting that the flat beta-strand of the Ile(5) stretch primed for self-assembly is a low-energy conformation of the Ile(5) stretch and rationalizing its high propensity for self-assembly. The ability of the PSAM to "solubilize" an otherwise insoluble peptide stretch suggests the potential of the PSAM approach to the characterization of self-assembling peptides.
Journal of Molecular Biology 05/2008; 378(2):459-67. · 4.00 Impact Factor
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ABSTRACT: β-Amyloid (Aβ) aggregates at low concentrations in vivo, and this may involve covalently modified forms of these peptides. Modification of Aβ by 4-hydroxynonenal (4-HNE) initially
increases the hydrophobicity of these peptides and subsequently leads to additional reactions, such as peptide cross-linking.
To model these initial events, without confounding effects of subsequent reactions, we modified Aβ at each of its amino groups
using a chemically simpler, close analogue of 4-HNE, the octanoyl group: K16-octanoic acid (OA)-Aβ, K28-OA-Aβ, and Nα-OA-Aβ.
Octanoylation of these sites on Aβ-(1–40) had strikingly different effects on fibril formation. K16-OA-Aβ and K28-OA-Aβ, but
not Nα-OA-Aβ, had increased propensity to aggregate. The type of aggregate (electron microscopic appearance) differed with
the site of modification. The ability of octanoyl-Aβ peptides to cross-seed solutions of Aβ was the inverse of their ability
to form fibrils on their own (i.e. Aβ ≈ Nα-OA-Aβ >> K16-OA-Aβ >> K28-OA-Aβ). By CD spectroscopy, K16-OA-Aβ and K28-OA-Aβ had increased β-sheet propensity compared
with Aβ-(1–40) or Nα-OA-Aβ. K16-OA-Aβ and K28-OA-Aβ were more amphiphilic than Aβ-(1–40) or Nα-OA-Aβ, as shown by lower “critical
micelle concentrations” and higher monolayer collapse pressures. Finally, K16-OA-Aβ and K28-OA-Aβ are much more cytotoxic
to N2A cells than Aβ-(1–40) or Nα-OA-Aβ. The greater cytotoxicity of K16-OA-Aβ and K28-OA-Aβ may reflect their greater amphiphilicity.
We conclude that lipidation can make Aβ more prone to aggregation and more cytotoxic, but these effects are highly site-specific.
Journal of Biological Chemistry 12/2007; 282(51):36987-36997. · 4.77 Impact Factor
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ABSTRACT: Beta-amyloid (Abeta) aggregates at low concentrations in vivo, and this may involve covalently modified forms of these peptides. Modification of Abeta by 4-hydroxynonenal (4-HNE) initially increases the hydrophobicity of these peptides and subsequently leads to additional reactions, such as peptide cross-linking. To model these initial events, without confounding effects of subsequent reactions, we modified Abeta at each of its amino groups using a chemically simpler, close analogue of 4-HNE, the octanoyl group: K16-octanoic acid (OA)-Abeta, K28-OA-Abeta, and Nalpha-OA-Abeta. Octanoylation of these sites on Abeta-(1-40) had strikingly different effects on fibril formation. K16-OA-Abeta and K28-OA-Abeta, but not Nalpha-OA-Abeta, had increased propensity to aggregate. The type of aggregate (electron microscopic appearance) differed with the site of modification. The ability of octanoyl-Abeta peptides to cross-seed solutions of Abeta was the inverse of their ability to form fibrils on their own (i.e. Abeta approximately Nalpha-OA-Abeta>K16-OA-Abeta>K28-OA-Abeta). By CD spectroscopy, K16-OA-Abeta and K28-OA-Abeta had increased beta-sheet propensity compared with Abeta-(1-40) or Nalpha-OA-Abeta. K16-OA-Abeta and K28-OA-Abeta were more amphiphilic than Abeta-(1-40) or Nalpha-OA-Abeta, as shown by lower "critical micelle concentrations" and higher monolayer collapse pressures. Finally, K16-OA-Abeta and K28-OA-Abeta are much more cytotoxic to N2A cells than Abeta-(1-40) or Nalpha-OA-Abeta. The greater cytotoxicity of K16-OA-Abeta and K28-OA-Abeta may reflect their greater amphiphilicity. We conclude that lipidation can make Abeta more prone to aggregation and more cytotoxic, but these effects are highly site-specific.
Journal of Biological Chemistry 12/2007; 282(51):36987-97. · 4.77 Impact Factor
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ABSTRACT: Polyglutamine (poly(Q)) expansion is associated with protein aggregation into beta-sheet amyloid fibrils and neuronal cytotoxicity. In the mutant poly(Q) protein huntingtin, associated with Huntington's disease, both aggregation and cytotoxicity may be abrogated by a polyproline (poly(P)) domain flanking the C terminus of the poly(Q) region. To understand structural changes that may occur with the addition of the poly(P) sequence, we synthesized poly(Q) peptides with 3-15 glutamine residues and a corresponding set of poly(Q) peptides flanked on the C terminus by 11 proline residues (poly(Q)-poly(P)), as occurs in the huntingtin sequence. The shorter soluble poly(Q) peptides (three or six glutamine residues) showed polyproline type II-like (PPII)-like helix conformation when examined by circular dichroism spectroscopy and were monomers as judged by size-exclusion chromatography (SEC), while the longer poly(Q) peptides (nine or 15 glutamine residues) showed a beta-sheet conformation by CD and defined oligomers by SEC. Soluble poly(Q)-poly(P) peptides showed PPII-like content but SEC showed poorly defined, overlapping oligomeric peaks, and as judged by CD these peptides retained significant PPII-like structure with increasing poly(Q) length. More importantly, addition of the poly(P) domain increased the threshold for fibril formation to approximately 15 glutamine residues. X-ray diffraction, electron microscopy, and film CD showed that, while poly(Q) peptides with >or=6 glutamine residues formed beta-sheet-rich fibrils, only the longest poly(Q)-poly(P) peptide (15 glutamine residues) did so. From these and other observations, we propose that poly(Q) domains exist in a "tug-of-war" between two conformations, a PPII-like helix and a beta-sheet, while the poly(P) domain is conformationally constrained into a proline type II helix (PPII). Addition of poly(P) to the C terminus of a poly(Q) domain induces a PPII-like structure, which opposes the aggregation-prone beta-sheet. These structural observations may shed light on the threshold phenomenon of poly(Q) aggregation, and support the hypothesized evolution of "protective" poly(P) tracts adjacent to poly(Q) aggregation domains.
Journal of Molecular Biology 12/2007; 374(3):688-704. · 4.00 Impact Factor
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Hookang Im,
Marika Manolopoulou,
Enrico Malito,
Yuequan Shen,
Ji Zhao,
Marie Neant-Fery,
Ching-Yu Sun, Stephen C Meredith,
Sangram S Sisodia,
Malcolm A Leissring,
Wei-Jen Tang
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ABSTRACT: Insulin-degrading enzyme (IDE) is a zinc metalloprotease that hydrolyzes amyloid-beta (Abeta) and insulin, which are peptides associated with Alzheimer disease (AD) and diabetes, respectively. Our previous structural analysis of substrate-bound human 113-kDa IDE reveals that the N- and C-terminal domains of IDE, IDE-N and IDE-C, make substantial contact to form an enclosed catalytic chamber to entrap its substrates. Furthermore, IDE undergoes a switch between the closed and open conformations for catalysis. Here we report a substrate-free IDE structure in its closed conformation, revealing the molecular details of the active conformation of the catalytic site of IDE and new insights as to how the closed conformation of IDE may be kept in its resting, inactive conformation. We also show that Abeta is degraded more efficiently by IDE carrying destabilizing mutations at the interface of IDE-N and IDE-C (D426C and K899C), resulting in an increase in Vmax with only minimal changes to Km. Because ATP is known to activate the ability of IDE to degrade short peptides, we investigated the interaction between ATP and activating mutations. We found that these mutations rendered IDE less sensitive to ATP activation, suggesting that ATP might facilitate the transition from the closed state to the open conformation. Consistent with this notion, we found that ATP induced an increase in hydrodynamic radius, a shift in electrophoretic mobility, and changes in secondary structure. Together, our results highlight the importance of the closed conformation for regulating the activity of IDE and provide new molecular details that will facilitate the development of activators and inhibitors of IDE.
Journal of Biological Chemistry 09/2007; 282(35):25453-63. · 4.77 Impact Factor
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ABSTRACT: The early stages of peptide and protein aggregation include the formation of soluble oligomers, some of which may be cytotoxic. There is a paucity of structural information on these oligomers, however, because they are temporally unstable and tend to aggregate further into insoluble protofibrils and fibrils. To obtain structural information on soluble oligomers, we have developed a procedure for encapsulating a fibril-forming peptide, Peptide 1 (NH2-SDDYYYGFGSNKFGRPRDD-COOH), in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine single bilayer vesicles (POPC SBVs). We also encapsulated a non-fibril forming peptide, Peptide 2 (NH2-EEWEE-COOH), in POPC SBVs. The nominal concentration of Peptide 1 in the resulting 40 nm diameter SBVs was 2.4 +/- 0.1 mM, well above the concentration at which Peptide 1 forms fibrils. We demonstrated that these peptides had indeed been encapsulated by measuring longitudinal relaxation times (T1) in the presence and absence of a paramagnetic substance, 1 mM Gd-EDTA, by NMR spectroscopy. When the peptides were free in solution, they showed the expected shortening of T1 times and broadening of NMR peaks. In contrast, peptide encapsulated in POPC SBVs were shielded from the effects of Gd-EDTA and showed preservation of T1 values and NMR line widths. To demonstrate that encapsulation inhibits fibril formation, we measured one-dimensional proton (1D-1H) NMR spectra of the peptides in solution, and of the encapsulated peptides immediately after encapsulation, and 4 days after encapsulation, because Peptide 1 forms fibrils within 1 day. A 2.8 mM solution of Peptide 1 shows the loss of NMR signal expected for a fibrillizing peptide. In contrast, the 1D-1H spectra of encapsulated Peptide 1 measured immediately after encapsulation and 4 days after encapsulation were essentially identical, with preservation of line width at 4 days, i.e., well within the time frame of most high-resolution NMR experiments. Encapsulation may provide a means to obtain high-resolution NMR data on unstable soluble oligomers of peptides implicated in amyloidoses such as Alzheimer's Disease and provide the first detailed structural information about these possibly cytotoxic species that have hitherto been inaccessible to analysis.
Journal of the American Chemical Society 01/2007; 128(51):16460-1. · 9.91 Impact Factor
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ABSTRACT: Monoclonal antibodies have become important therapeutic agents against certain cancers. Many tumor-specific antigens are mutant proteins that are predominantly intracellular and thus not readily accessible to monoclonal antibodies. We found that a wild-type transmembrane protein could be transformed into a tumor-specific antigen. A somatic mutation in the chaperone gene Cosmc abolished function of a glycosyltransferase, disrupting O-glycan Core 1 synthesis and creating a tumor-specific glycopeptidic neo-epitope consisting of a monosaccharide and a specific wild-type protein sequence. This epitope induced a high-affinity, highly specific, syngeneic monoclonal antibody with antitumor activity. Such tumor-specific glycopeptidic neo-epitopes represent potential targets for monoclonal antibody therapy.
Science 11/2006; 314(5797):304-8. · 31.20 Impact Factor
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ABSTRACT: In a recent model of beta-amyloid (Abeta) fibrils, based mainly on solid-state NMR data, a molecular layer consists of two beta-sheets (residues 12-23 and 31-40 of Abeta1-40), folded onto one another by a connecting "bend" structure (residues 25-29) in the side-chain dimension. In this paper, we use two N-methyl amino acids to disrupt each of the two beta-sheets individually (2NMe(NTerm), residues 17 and 19; and 2NMe(CTerm), residues 37 and 39), or both of them at the same time (4NMe, with the above four N-methylated residues). Our data indicate that incorporation of two N-methyl amino acids into one beta-sheet is sufficient to disrupt that sheet while leaving the other, unmodified beta-sheet intact and able to form fibrils. We show, however, that disruption of each of the two beta-sheets has strikingly different effects on fibrillogenesis kinetics and fibril morphology. Both 2NMe(NTerm) and 2NMe(CTerm) form fibrils at similar rates, but more slowly than that of unmodified Abeta1-40. Electron microscopy shows that 2NMe(NTerm) forms straight fibrils with fuzzy amorphous material coating the edges, while 2NMe(CTerm) forms very regular, highly twisted fibrils-in both cases, distinct from the morphology of Abeta1-40 fibrils. Both 2NMe peptides show a "CMC" approximately four times greater than that of Abeta1-40. CD spectra of these peptides also evolve differently in time: whereas the CD spectra of 2NMe(NTerm) evolve little over 10 days, those of 2NMe(CTerm) show a transition to high beta-sheet content at about day 4-5. We also show that disruption of both beta-sheet domains, as in 4NMe, prevents fibril formation altogether, and renders Abeta1-40 highly water soluble and monomeric, and with solvent-exposed side chains. In summary, our data show (1) that the two beta-sheet domains fold in a semiautonomous manner, since disrupting each one still allows the other to fold; (2) that disruption of the N-terminal beta-sheet has a more profound effect on fibrillogenesis than disruption of the C-terminal beta-sheet, suggesting that the former is the more critical for the overall structure of the fibril; and (3) that disruption of both beta-sheet domains renders the peptide monomeric and unable to form fibrils.
Biochemistry 09/2006; 45(31):9485-95. · 3.42 Impact Factor
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STEPHEN C. MEREDITH
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ABSTRACT: Protein aggregation is a prominent feature of many neurodegenerative diseases, such as Alzheimer's, Huntington's, and Parkinson's diseases, as well as spongiform encephalopathies and systemic amyloidoses. These diseases are sometimes called protein misfolding diseases, but the latter term begs the question of what is the “folded” state of proteins for which normal structure and function are unknown. Amyloid consists of linear, unbranched protein or peptide fibrils of ∼100 Å diameter. These fibrils are composed of a wide variety of proteins that have no sequence homology, and no similarity in three-dimensional structures—and yet, as fibrils, they share a common secondary structure, the β-sheet. Because of the prominence of amyloid deposits in many of these diseases, much effort has gone into elucidation of fibril structure. Recent advances in solid-state NMR spectroscopy and other biophysical techniques have led to the partial elucidation of fibril structure. Surprisingly at the time, for β-amyloid, a set of 39–43-amino-acid peptides believed to play a pathogenic role in Alzheimer's disease, the β-sheets are parallel with all amino acids of the sheets in-register. Since the time of those observations, however, it has become clear that there is no universal structure for amyloid fibrils. While many of the amyloid fibrils described thus far have a parallel β-sheet structure, some have antiparallel β-sheets, and other, more subtle structural differences among amyloids exist as well. Amyloids demonstrate conformational plasticity, the ability to adopt more than one stable tertiary fold. Conformational plasticity could account for “strain” differences in prions, and for the fact that a single polypeptide can form different fibril types with conformational differences at the atomic level.More recent data now indicate that the fibrils may not be the most potent or proximate mediators of cyto- and neurotoxicity. This damage is not confined to cell death, but also includes more subtle forms of damage, such as disruption of synaptic plasticity in the central nervous system. Rather than fibrils, prefibrillar aggregates, variously called “micelles,”“protofibrils,” or ADDLs (β-amyloid-derived diffusible ligands in the case of β-amyloid) may be the more proximate mediators of cell damage. These are soluble oligomers of aggregating peptides or proteins, but their structure is very challenging to study, because they are generally difficult to obtain in large enough quantities for high-resolution structural techniques, and they are temporally unstable, rapidly changing into more mature, and eventually fibrillar forms. Consequently, the mechanisms by which they disrupt cellular function are also not well understood. Nevertheless, three broad, overlapping, nonexclusive sets of mechanisms have been proposed as responsible for the cellular damage caused by soluble, oligomeric protein aggregates. These are: (1) disruption of cell membranes and their functions [e.g., by inserting into membranes and disrupting normal ion gradients]; (2) inactivation of normally folded, functional proteins [e.g., by sequestering or localizing transcription factors to the wrong cellular compartment]; and (3) “gumming up the works,” by binding to and inactivating components of the quality-control system of cells, such as the proteasome or chaperone proteins.
Annals of the New York Academy of Sciences 02/2006; 1066(1):181 - 221. · 3.15 Impact Factor
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ABSTRACT: This review considers the design, synthesis, and mechanistic assessment of peptide-based fibrillogenesis inhibitors, mainly focusing on beta-amyloid, but generalizable to other aggregating proteins and peptides. In spite of revision of the "amyloid hypothesis," the investigation and development of fibrillogenesis inhibitors remain important scientific and therapeutic goals for at least three reasons. First, it is still premature to dismiss fibrils altogether as sources of cytotoxicity. Second, a "fibrillogenesis inhibitor" is typically identified experimentally as such, but these compounds may also bind to intermediates in the fibrillogenesis pathway and have hard-to-predict consequences, including improved clearance of more cytotoxic soluble oligomers. Third, inhibitors are valuable structural probes, as the entire field of enzymology attests. Screening procedures for selection of random inhibitory sequences are briefly considered, but the bulk of the review concentrates on rationally designed fibrillogenesis inhibitors. Among these are internal segments of fibril-forming peptides, amino acid substitutions and side chain modifications of fibrillogenic domains, insertion of prolines into or adjacent to fibrillogenic domains, modification of peptide termini, modification of peptide backbone atoms (including N-methylation), peptide cyclization, use of D-amino acids in fibrillogenic domains, and nonpeptidic beta-sheet mimics. Finally, we consider methods of assaying fibrillogenesis inhibitors, including pitfalls in these assays. We consider binding of inhibitor peptides to their targets, but because this is a specific application of the more general and much larger problem of assessing protein-protein interactions, this topic is covered only briefly. Finally, we consider potential applications of inhibitor peptides to therapeutic strategies.
Methods in Enzymology 02/2006; 413:273-312. · 2.04 Impact Factor
