Protein folding and disease: a view from the first Horizon Symposium.
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Alzheimer Disease↗
Biomedical subjects
Christopher M Dobson
Publications and source records attributed to Christopher M Dobson.
Superficial granulomatous pyoderma treated with intravenous immunoglobulin.
Superficial granulomatous pyoderma is a rare variant of pyoderma gangrenosum. The superficial ulceration and vegetative margins are clinically distinctive; the suppurative and granulomatous histology is characteristic, though not pathognomonic. The condition is said to be relatively benign and more responsive to treatment than classic pyoderma gangrenosum, though published evidence of this is not always convincing. We present a case of superficial granulomatous pyoderma that was unusually aggressive, but that underwent dramatic and lasting resolution after intravenous immunoglobulin therapy.
Aged↗
Telangiectatic metastatic breast carcinoma in face and scalp mimicking cutaneous angiosarcoma.
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Breast Neoplasms↗
Structural dissection of alkaline-denatured pepsin.
It has been established in a number of studies that the alkaline-denatured state of pepsin (the I(P) state) is composed of a compact C-terminal lobe and a largely unstructured N-terminal lobe. In the present study, we have investigated the residual structure in the I(P) state in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C-terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N-terminal lobe contributes a substantial amount of additional residual structure to the I(P) state of pepsin. CD spectra indicate in addition that significant nonnative alpha-helical structure is present in the C-terminal lobe of the structure when the N-terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the I(P) state is significantly more complex than that of a fully folded C-terminal lobe connected to an unstructured N-terminal lobe.
Amino Acid Sequence↗
Chemical modification of insulin in amyloid fibrils.
We have investigated the chemical modification of insulin under conditions that promote the conversion of the soluble protein into amyloid fibrils. The modifications that are incorporated into the fibrils include deamidation of Asn A21, Asn B3, and Gln B4. In order to prepare fibrils with minimal deamidation of these residues, the kinetics of aggregation were accelerated by seeding with aliquots of a solution containing preformed fibrils. The resulting fibrils were then reincubated to determine the extent to which chemical modification occurs in the fibril itself. The deamidation of Asn A21 in particular could be followed in detail. Deamidation of this residue in the fibrillar form of insulin was found to occur in only 52 +/- 5% of molecules. This result indicates that there are at least two different packing environments of insulin molecules in the fibrils and suggests that the characterization of chemical modifications may be a useful probe of the environment of polypeptide chains within amyloid fibrils.
Amino Acid Sequence↗
Molecular conformation of a peptide fragment of transthyretin in an amyloid fibril.
The molecular conformation of peptide fragment 105-115 of transthyretin, TTR(105-115), previously shown to form amyloid fibrils in vitro, has been determined by magic-angle spinning solid-state NMR spectroscopy. 13C and 15N linewidth measurements indicate that TTR(105-115) forms a highly ordered structure with each amino acid in a unique environment. 2D 13C-13C and 15N-13C-13C chemical shift correlation experiments, performed on three fibril samples uniformly 13C,15N-labeled in consecutive stretches of 4 aa, allowed the complete sequence-specific backbone and side-chain 13C and 15N resonance assignments to be obtained for residues 105-114. Analysis of the 15N, 13CO, 13Calpha, and 13Cbeta chemical shifts allowed quantitative predictions to be made for the backbone torsion angles phi and psi. Furthermore, four backbone 13C-15N distances were determined in two selectively 13C,15N-labeled fibril samples by using rotational-echo double-resonance NMR. The results show that TTR(105-115) adopts an extended beta-strand conformation that is similar to that found in the native protein except for substantial differences in the vicinity of the proline residue.
Amino Acid Sequence↗
De novo designed peptide-based amyloid fibrils.
Identification of therapeutic strategies to prevent or cure diseases associated with amyloid fibril deposition in tissue (Alzheimer's disease, spongiform encephalopathies, etc.) requires a rational understanding of the driving forces involved in the formation of these organized assemblies rich in beta-sheet structure. To this end, we used a computer-designed algorithm to search for hexapeptide sequences with a high propensity to form homopolymeric beta-sheets. Sequences predicted to be highly favorable on this basis were found experimentally to self-associate efficiently into beta-sheets, whereas point mutations predicted to be unfavorable for this structure inhibited polymerization. However, the property to form polymeric beta-sheets is not a sufficient requirement for fibril formation because, under the conditions used here, preformed beta-sheets from these peptides with charged residues form well defined fibrils only if the total net charge of the molecule is +/-1. This finding illustrates the delicate balance of interactions involved in the formation of fibrils relative to more disordered aggregates. The present results, in conjunction with x-ray fiber diffraction, electron microscopy, and Fourier transform infrared measurements, have allowed us to propose a detailed structural model of the fibrils.
Algorithms↗
Determination of a transition state at atomic resolution from protein engineering data.
We present a method for determining the structure of the transition state ensemble (TSE) of a protein by using phi values derived from protein engineering experiments as restraints in molecular dynamics simulations employing a realistic all-atom molecular mechanics energy function. The method uses a biasing potential to select an ensemble of structures having phi values in agreement with the experimental data set. An application to acylphosphatase (AcP), a protein for which phi values have been measured for 24 out of 98 residues, illustrates the approach. The properties of the TSE determined in this way are compared with those of a coarse-grained model obtained using a Monte Carlo (MC) sampling method based on a C(alpha) representation of the structure. The two TSEs determined at different structural resolution are consistent and complementary. While the C(alpha) model allows better sampling of the conformation space occupied by the transition state, the all-atom model offers a more detailed description of the structural and energetic properties of the conformations included in the TSE. The combination of low-resolution C(alpha) results with all-atom molecular dynamics simulations provides a powerful and general method for determining the nature of TSEs from protein engineering data.
Acid Anhydride Hydrolases↗
Altered aggregation properties of mutant gamma-crystallins cause inherited cataract.
Protein inclusions are associated with a diverse group of human diseases ranging from localized neurological disorders through to systemic non-neuropathic diseases. Here, we present evidence that the formation of intranuclear inclusions is a key event in cataract formation involving altered gamma-crystallins that are un likely to adopt their native fold. In three different inherited murine cataracts involving this type of gamma-crystallin mutation, large inclusions containing the altered gamma-crystallins were found in the nuclei of the primary lens fibre cells. Their formation preceded not only the first gross morphological changes in the lens, but also the first signs of cataract. The inclusions contained filamentous material that could be stained with the amyloid-detecting dye, Congo red. In vitro, recombinant mutant gammaB-crystallin readily formed amyloid fibrils under physiological buffer conditions, unlike wild-type protein. These data suggest that this type of cataract is caused by a mechanism involving the nuclear targeting and deposition of amyloid-like inclusions. The mutant gamma-crystallins initially disrupt nuclear function, but then this progresses to a full cataract phenotype.
Amyloid↗
Probing the exposure of tyrosine and tryptophan residues in partially folded proteins and folding intermediates by CIDNP pulse-labeling.
A nuclear magnetic resonance (NMR) technique has been devised to probe the structures of disordered, partially folded states of proteins at the level of individual amino acid residues. Chemically induced dynamic nuclear polarization (CIDNP) is first generated in exposed aromatic side-chains of the denatured state and then transferred to the high-resolution NMR spectrum of the native state by stimulating rapid refolding of the protein. Crucial improvements in sensitivity were achieved by carrying out the polarization-producing photochemistry in a deoxygenated sample of the disordered state of the protein in a magnetic field of 4.0 T and recording the (1)H NMR spectrum of the refolded native state at 9.4 T (400 MHz). Application of this method to the low pH molten-globule state of alpha-lactalbumin reveals remarkably nativelike environments for the aromatic residues in the primary hydrophobic core of the protein. This result provides compelling evidence that the detailed fold of a protein can be established prior to the formation of the cooperative close-packed native structure.
Lactalbumin↗
The behaviour of polyamino acids reveals an inverse side chain effect in amyloid structure formation.
Amyloid fibrils and prions are proteinaceous aggregates that are based on a unique form of polypeptide configuration, termed cross-beta structure. Using a group of chemically distinct polyamino acids, we show here that the existence of such a structure does not require the presence of specific side chain interactions or sequence patterns. These observations firmly establish that amyloid formation and protein folding represent two fundamentally different ways of organizing polypeptides into ordered conformations. Protein folding depends critically on the presence of distinctive side chain sequences and produces a unique globular fold. By contrast, the properties of different polyamino acids suggest that amyloid formation arises primarily from main chain interactions that are, in some environments, overruled by specific side chain contacts. This side chain effect can be thought of as the inverse of the one that characterizes protein folding. Conditions including Alzheimer's and Creutzfeldt-Jakob diseases represent, on this basis, pathological cases in which a natural polypeptide chain has aberrantly adopted the conformation that is primarily defined by main chain interactions and not the structure that is determined by specific side chain contacts that depend on the polypeptide sequence.
Amino Acids↗
Stimulation and inhibition of fibril formation by a peptide in the presence of different concentrations of SDS.
Sodium dodecyl sulphate (SDS), a detergent that mimics some characteristics of biological membranes, has been found to affect significantly fibril formation by a peptide from human complement receptor 1. In aqueous solution the peptide is unfolded but slowly aggregates to form fibrils. In sub-micellar concentrations of SDS the peptide is initially alpha-helical but converts rapidly to a beta-sheet structure and large quantities of fibrils form. In SDS above the critical micellar concentration the peptide adopts a stable alpha-helical structure and no fibrils are observed. These findings demonstrate the sensitivity of fibril formation to solution conditions and suggest a possible role for membrane components in amyloid fibril formation in living systems.
Circular Dichroism↗
Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases.
Protein aggregation and the formation of highly insoluble amyloid structures is associated with a range of debilitating human conditions, which include Alzheimer's disease, Parkinson's disease, and the Creutzfeldt-Jakob disease. Muscle acylphosphatase (AcP) has already provided significant insights into mutational changes that modulate amyloid formation. In the present paper, we have used this system to investigate the effects of mutations that modify the charge state of a protein without affecting significantly the hydrophobicity or secondary structural propensities of the polypeptide chain. A highly significant inverse correlation was found to exist between the rates of aggregation of the protein variants under denaturing conditions and their overall net charge. This result indicates that aggregation is generally favored by mutations that bring the net charge of the protein closer to neutrality. In light of this finding, we have analyzed natural mutations associated with familial forms of amyloid diseases that involve alteration of the net charge of the proteins or protein fragments associated with the diseases. Sixteen mutations have been identified for which the mechanism of action that causes the pathological condition is not yet known or fully understood. Remarkably, 14 of these 16 mutations cause the net charge of the corresponding peptide or protein that converts into amyloid deposits to be reduced. This result suggests that charge has been a key parameter in molecular evolution to ensure the avoidance of protein aggregation and identifies reduction of the net charge as an important determinant in at least some forms of protein deposition diseases.
Alzheimer Disease↗
Getting out of shape.
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Alzheimer Disease↗
The protofilament structure of insulin amyloid fibrils.
Under solution conditions where the native state is destabilized, the largely helical polypeptide hormone insulin readily aggregates to form amyloid fibrils with a characteristic cross-beta structure. However, there is a lack of information relating the 4.8 A beta-strand repeat to the higher order assembly of amyloid fibrils. We have used cryo-electron microscopy (EM), combining single particle analysis and helical reconstruction, to characterize these fibrils and to study the three-dimensional (3D) arrangement of their component protofilaments. Low-resolution 3D structures of fibrils containing 2, 4, and 6 protofilaments reveal a characteristic, compact shape of the insulin protofilament. Considerations of protofilament packing indicate that the cross-beta ribbon is composed of relatively flat beta-sheets rather than being the highly twisted, beta-coil structure previously suggested by analysis of globular protein folds. Comparison of the various fibril structures suggests that very small, local changes in beta-sheet twist are important in establishing the long-range coiling of the protofilaments into fibrils of diverse morphology.
Amyloid↗
The interaction of the molecular chaperone alpha-crystallin with unfolding alpha-lactalbumin: a structural and kinetic spectroscopic study.
The unfolding of the apo and holo forms of bovine alpha-lactalbumin (alpha-LA) upon reduction by dithiothreitol (DTT) in the presence of the small heat-shock protein alpha-crystallin, a molecular chaperone, has been monitored by visible and UV absorption spectroscopy, mass spectrometry and (1)H NMR spectroscopy. From these data, a description and a time-course of the events that result from the unfolding of both forms of the protein, and the state of the protein that interacts with alpha-crystallin, have been obtained. alpha-LA contains four disulphide bonds and binds a calcium ion. In apo alpha-LA, the disulphide bonds are reduced completely over a period of approximately 1500 seconds. Fully reduced alpha-LA adopts a partly folded, molten globule conformation that aggregates and, ultimately, precipitates. In the presence of an equivalent mass of alpha-crystallin, this precipitation can be prevented via complexation with the chaperone. alpha-Crystallin does not interfere with the kinetics of the reduction of disulphide bonds in apo alpha-LA but does stabilise the molten globule state. In holo alpha-LA, the disulphide bonds are less accessible to DTT, because of the stabilisation of the protein by the bound calcium ion, and reduction occurs much more slowly. A two-disulphide intermediate aggregates and precipitates rapidly. Its precipitation can be prevented only in the presence of a 12-fold mass excess of alpha-crystallin. It is concluded that kinetic factors are important in determining the efficiency of the chaperone action of alpha-crystallin. It interacts efficiently with slowly aggregating, highly disordered intermediate (molten globule) states of alpha-LA. Real-time NMR spectroscopy shows that the kinetics of the refolding of apo alpha-LA following dilution from denaturant are not affected by the presence of alpha-crystallin. Thus, alpha-crystallin is not a chaperone that is involved in protein folding per se. Rather, its role is to stabilise compromised, partly folded, molten globule states of proteins that are destined for precipitation.
Animals↗
Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.
A range of human degenerative conditions, including Alzheimer's disease, light-chain amyloidosis and the spongiform encephalopathies, is associated with the deposition in tissue of proteinaceous aggregates known as amyloid fibrils or plaques. It has been shown previously that fibrillar aggregates that are closely similar to those associated with clinical amyloidoses can be formed in vitro from proteins not connected with these diseases, including the SH3 domain from bovine phosphatidyl-inositol-3'-kinase and the amino-terminal domain of the Escherichia coli HypF protein. Here we show that species formed early in the aggregation of these non-disease-associated proteins can be inherently highly cytotoxic. This finding provides added evidence that avoidance of protein aggregation is crucial for the preservation of biological function and suggests common features in the origins of this family of protein deposition diseases.
3T3 Cells↗
Long-range interactions within a nonnative protein.
Protein folding and unfolding are coupled to a range of biological phenomena, from the regulation of cellular activity to the onset of neurodegenerative diseases. Defining the nature of the conformations sampled in nonnative proteins is crucial for understanding the origins of such phenomena. We have used a combination of nuclear magnetic resonance (NMR) spectroscopy and site-directed mutagenesis to study unfolded states of the protein lysozyme. Extensive clusters of hydrophobic structure exist within the wild-type protein even under strongly denaturing conditions. These clusters involve distinct regions of the sequence but are all disrupted by a single point mutation that replaced residue Trp62 with Gly located at the interface of the two major structural domains in the native state. Thus, nativelike structure in the denatured protein is stabilized by the involvement of Trp62 in nonnative and long-range interactions.
Amino Acid Substitution↗