PubMed Health⌕ Search

Biomedical subjects

Evan T Powers

Publications and source records attributed to Evan T Powers.

At least 19 recordsLinked to original sources
PubMed2006

The kinetics of nucleated polymerizations at high concentrations: amyloid fibril formation near and above the "supercritical concentration".

The formation of amyloid and other types of protein fibrils is thought to proceed by a nucleated polymerization mechanism. One of the most important features commonly associated with nucleated polymerizations is a strong dependence of the rate on the concentration. However, the dependence of fibril formation rates on concentration can weaken and nearly disappear as the concentration increases. Using numerical solutions to the rate equations for nucleated polymerization and analytical solutions to some limiting cases, we examine this phenomenon and show that it is caused by the concentration approaching and then exceeding the equilibrium constant for dissociation of monomers from species smaller than the nucleus, a quantity we have named the "supercritical concentration". When the concentration exceeds the supercritical concentration, the monomer, not the nucleus, is the highest-energy species on the fibril formation pathway, and the fibril formation reaction behaves initially like an irreversible polymerization. We also derive a relation that can be used in a straightforward method for determining the nucleus size and the supercritical concentration from experimental measurements of fibril formation rates.

Amyloid↗
PubMed2006

Elevated levels of oxidized cholesterol metabolites in Lewy body disease brains accelerate alpha-synuclein fibrilization.

Oxidative stress, inflammation and alpha-synuclein overexpression confer risk for development of alpha-synucleinopathies-neurodegenerative diseases that include Parkinson disease and Lewy body dementia. Dopaminergic neurons undergo degeneration in these diseases and are particularly susceptible to oxidative stress because dopamine metabolism itself creates reactive oxygen species. Intraneuronal deposition of alpha-synuclein as amyloid fibrils or Lewy bodies is the hallmark of these diseases. Herein, we demonstrate that concentrations of oxidative cholesterol metabolites derived from reactive oxygen species are elevated in the cortices of individuals with Lewy body dementia relative to those of age-matched controls, and we show that these metabolites accelerate alpha-synuclein aggregation in vitro. The increase in the production of these cytotoxic cholesterol metabolites is also observed in a dopaminergic cell line that overexpresses alpha-synuclein. By extension, these data lead to the hypothesis that oxidative stress produces cholesterol aldehydes that enable alpha-synuclein aggregation, leading to a pathologic cycle.

Brain↗
PubMed2006

Beta-sheet folding mechanisms from perturbation energetics.

Amide backbone and sidechain mutagenesis data can be used in combination with kinetic and thermodynamic measurements to understand the energetic contributions of backbone hydrogen bonding and the hydrophobic effect to the acquisition of beta-sheet structure. For example, it has been revealed that loop 1 of the WW domain forms in the transition state, consistent with the emerging theme that reverse turn formation is rate limiting in beta-sheet folding. A distinct subset of WW domain residues principally influences thermodynamic stability by forming hydrogen bonds and hydrophobic interactions that stabilize the native state. Energetic data and sequence mining reveal that only a small subset of the molecular information contained in sequences or observed in high-resolution structures is required to generate folded functional beta-sheets, consistent with evolutionary robustness.

Animals↗
PubMed2006

Small molecule oxidation products trigger disease-associated protein misfolding.

Oxidative stress and inflammation are risk factors for both the development of alpha-synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies, and Alzheimer's disease, the two most common neurodegenerative disorders. These diseases are associated with the neurotoxic deposition of misassembled alpha-synuclein and amyloid-beta (Abeta) peptides, respectively. Both occur sporadically, that is, without detectable disease-related mutations, in the vast majority of cases. Small molecule oxidation products, especially secosterols derived from cholesterol and 4-hydroxynonenal derived from lipid peroxidation, found in afflicted brains, accelerate the misassembly of both Abeta and alpha-synuclein. This Account explores the mechanism of small molecule oxidation product-mediated protein misassembly and possible intervention strategies.

Aldehydes↗
PubMed2005

Partitioning conformational intermediates between competing refolding and aggregation pathways: insights into transthyretin amyloid disease.

Amyloid diseases are caused by the aberrant assembly of a protein in the extracellular space. Folded proteins are not amyloidogenic; however, the native state is generally in equilibrium with a minor population of unfolded or partially folded aggregation-competent conformers outside of the cell. Understanding how the partially unfolded conformers kinetically partition between the competing refolding and aggregation pathways provides insight into how misfolding, which occurs continuously, becomes pathogenic. Towards this end, we have previously studied the amyloidogenicity of transthyretin (TTR), a human beta-sheet-rich homotetrameric protein that must undergo rate-limiting tetramer dissociation and partial monomer unfolding to misassemble into amyloid and other aggregates. We demonstrate herein that TTR homotetramers reassemble by an unusual monomer-dimer-trimer-tetramer (MDRT) pathway. Therefore, the rate of every step in the reassembly pathway is dependent on the concentration of folded TTR monomer. Partitioning soluble TTR monomers between the reassembly pathway and the aggregation pathway should therefore depend on the relative concentrations of aggregates and assembly intermediates. Aggregate clearance is envisioned to play an important role in the partitioning of protein in vivo, where partitioning to the aggregation pathway becomes increasingly favorable under conditions where the concentration of aggregates is increased because aggregate clearance is slow relative to the rate of aggregation. This shift from efficient to inefficient aggregate clearance could occur with aging, offering an explanation for the age-associated nature of these neurodegenerative diseases.

Amyloidosis↗
PubMed2005

Controlling the morphology of cross beta-sheet assemblies by rational design.

Low molecular weight peptidomimetics with simple amphiphilic sequences can help to elucidate the structures of cross beta-sheet assemblies, such as amyloid fibrils. The peptidomimetics described herein comprise a dibenzofuran template, two peptide strands made up of alternating hydrophilic and hydrophobic residues, and carboxyl termini, each of which can be varied to probe the structural requirements for beta-sheet self-assembly processes. The dibenzofuran template positions the strands approximately 10 A apart, allowing corresponding hydrophobic side chains in the strands to pack into a collapsed U-shaped structure. This conformation is stabilized by hydrophobic interactions, not intramolecular hydrogen bonds. Intermolecular stacking of the collapsed peptidomimetics, enabled by intermolecular hydrogen bonding and hydrophobic interactions, affords 25-27 A wide protofilaments having a cross beta-sheet structure. Association of protofilaments, mediated by the dibenzofuran substructures and driven by the hydrophobic effect, affords 50-60 A wide filaments. These widths can be controlled by changing the length of the peptide strands. Further assembly of the filaments into fibrils or ribbons can be controlled by modification of the template, C-terminus, and buffer ion composition.

Amides↗
PubMed2005

Potent and selective structure-based dibenzofuran inhibitors of transthyretin amyloidogenesis: kinetic stabilization of the native state.

Transthyretin (TTR) amyloidogenesis requires rate-limiting tetramer dissociation and partial monomer denaturation to produce a misassembly competent species. This process has been followed by turbidity to identify transthyretin amyloidogenesis inhibitors including dibenzofuran-4,6-dicarboxylic acid (1). An X-ray cocrystal structure of TTR.1(2) reveals that it only utilizes the outer portion of the two thyroxine binding pockets to bind to and inhibit TTR amyloidogenesis. Herein, structure-based design was employed to append aryl substituents at C1 of the dibenzofuran ring to complement the unused inner portion of the thyroxine binding pockets. Twenty-eight amyloidogenesis inhibitors of increased potency and dramatically increased plasma TTR binding selectivity resulted. These function by imposing kinetic stabilization on the native tetrameric structure of TTR, creating a barrier that is insurmountable under physiological conditions. Since kinetic stabilization of the TTR native state by interallelic trans suppression is known to ameliorate disease, there is reason to be optimistic that the dibenzofuran-based inhibitors will do the same. Preventing the onset of amyloidogenesis is the most conservative strategy to intervene clinically, as it remains unclear which of the TTR misassembly intermediates results in toxicity. The exceptional binding selectivity enables these inhibitors to occupy the thyroxine binding site(s) in a complex biological fluid such as blood plasma, required for inhibition of amyloidogenesis in humans. It is now established that the dibenzofuran-based amyloidogenesis inhibitors have high selectivity, affinity, and efficacy and are thus excellent candidates for further pharmacologic evaluation.

Amyloid↗
PubMed2005

Oxidative metabolites accelerate Alzheimer's amyloidogenesis by a two-step mechanism, eliminating the requirement for nucleation.

The process of amyloid formation by the amyloid beta peptide (Abeta), i.e., the misassembly of Abetapeptides into soluble quaternary structures and, ultimately, amyloid fibrils, appears to be at the center of Alzheimer's disease (AD) pathology. We have shown that abnormal oxidative metabolites, including cholesterol-derived aldehydes, modify Abeta and accelerate the early stages of amyloidogenesis (the formation of spherical aggregates). This process, which we have termed metabolite-initiated protein misfolding, could explain why hypercholesterolemia and inflammation are risk factors for sporadic AD. Herein, the mechanism by which cholesterol metabolites hasten Abeta 1-40 amyloidogenesis is explored, revealing a process that has at least two steps. In the first step, metabolites modify Abeta peptides by Schiff base formation. The Abeta-metabolite adducts form spherical aggregates by a downhill polymerization that does not require a nucleation step, dramatically accelerating Abeta aggregation. In agitated samples, a second step occurs in which fibrillar aggregates form, a step also accelerated by cholesterol metabolites. However, the metabolites do not affect the rate of fibril growth in seeded aggregation assays; their role appears to be in initiating amyloidogenesis by lowering the critical concentration for aggregation into the nanomolar range. Small molecules that block Schiff base formation inhibit the metabolite effect, demonstrating the importance of the covalent adduct. Metabolite-initiated amyloidogenesis offers an explanation for how Abeta aggregation could occur at physiological nanomolar concentrations.

Alzheimer Disease↗
PubMed2005

Design, synthesis, and evaluation of oxazole transthyretin amyloidogenesis inhibitors.

Ten oxazoles bearing a C(4) carboxyl group were synthesized and evaluated as transthyretin (TTR) amyloid fibril inhibitors. Substituting aryls at the C(2) position of the oxazole ring reveals that a 3,5-dichlorophenyl substituent significantly reduced amyloidogenesis. The efficacy of these inhibitors was enhanced further by installing an ethyl, a propyl, or a CF(3) group at the C(5) position. The CF(3) substitution at C(5) also improves the TTR binding selectivity over all the other proteins in human blood.

Amyloid↗
PubMed2005

Backbone-Backbone H-Bonds Make Context-Dependent Contributions to Protein Folding Kinetics and Thermodynamics: Lessons from Amide-to-Ester Mutations.

The contribution of backbone-backbone hydrogen bonds (H-bonds) to protein folding energetics has been controversial. This is due, at least in part, to the inability to perturb backbone-backbone H-bonds by traditional methods of protein mutagenesis. Recently, however, protein backbone mutagenesis has become possible with the development of chemical and biological methods to replace individual amides in the protein backbone with esters. Here, we review the use of amide-to-ester mutation as a tool to evaluate the contribution of backbone-backbone H-bonds to protein folding kinetics and thermodynamics.

Amides↗
PubMed2005

Influence of the N-terminal domain on the aggregation properties of the prion protein.

Prion diseases appear to be caused by the aggregation of the cellular prion protein (PrP(C)) into an infectious form denoted PrP(Sc). The in vitro aggregation of the prion protein has been extensively investigated, yet many of these studies utilize truncated polypeptides. Because the C-terminal portion of PrP(Sc) is protease-resistant and retains infectivity, it is assumed that studies on this fragment are most relevant. The full-length protein can be distinguished from the truncated protein because it contains a largely structured, alpha-helical, C-terminal region in addition to an N terminus that is unstructured in the absence of metal ion binding. Herein, the in vitro aggregation of a truncated portion of the prion protein (PrP 90-231) and a full-length version (PrP 23-231) were compared. In each case, concentration-dependent aggregation was analyzed to discern whether it proceeds by a nucleation-dependent pathway. Both protein constructs appear to aggregate via a nucleated polymerization with a small nucleus size, yet the later steps differ. The full-length protein forms larger aggregates than the truncated protein, indicating that the N terminus may mediate higher-order aggregation processes. In addition, the N terminus has an influence on the assembly state of PrP before aggregation begins, causing the full-length protein to adopt several oligomeric forms in a neutral pH buffer. Our results emphasize the importance of studying the full-length protein in addition to the truncated forms for in vitro aggregation studies in order to make valid hypotheses about the mechanisms of prion aggregation and the distribution of aggregates in vivo.

Animals↗
PubMed2004

Context-dependent contributions of backbone hydrogen bonding to beta-sheet folding energetics.

Backbone hydrogen bonds (H-bonds) are prominent features of protein structures; however, their role in protein folding remains controversial because they cannot be selectively perturbed by traditional methods of protein mutagenesis. Here we have assessed the contribution of backbone H-bonds to the folding kinetics and thermodynamics of the PIN WW domain, a small beta-sheet protein, by individually replacing its backbone amides with esters. Amide-to-ester mutations site-specifically perturb backbone H-bonds in two ways: a H-bond donor is eliminated by replacing an amide NH with an ester oxygen, and a H-bond acceptor is weakened by replacing an amide carbonyl with an ester carbonyl. We perturbed the 11 backbone H-bonds of the PIN WW domain by synthesizing 19 amide-to-ester mutants. Thermodynamic studies on these variants show that the protein is most destabilized when H-bonds that are enveloped by a hydrophobic cluster are perturbed. Kinetic studies indicate that native-like secondary structure forms in one of the protein's loops in the folding transition state, but the backbone is less ordered elsewhere in the sequence. Collectively, our results provide an unusually detailed picture of the folding of a beta-sheet protein.

Amides↗
PubMed2004

Transthyretin aggregation under partially denaturing conditions is a downhill polymerization.

The deposition of fibrils and amorphous aggregates of transthyretin (TTR) in patient tissues is a hallmark of TTR amyloid disease, but the molecular details of amyloidogenesis are poorly understood. Tetramer dissociation is typically rate-limiting for TTR amyloid fibril formation, so we have used a monomeric variant of TTR (M-TTR) to study the mechanism of aggregation. Amyloid formation is often considered to be a nucleation-dependent process, where fibril growth requires the formation of an oligomeric nucleus that is the highest energy species on the pathway. According to this model, the rate of fibril formation should be accelerated by the addition of preformed aggregates or "seeds", which effectively bypasses the nucleation step. Herein, we demonstrate that M-TTR amyloidogenesis at low pH is a complex, multistep reaction whose kinetic behavior is incompatible with the expectations for a nucleation-dependent polymerization. M-TTR aggregation is not accelerated by seeding, and the dependence of the reaction timecourse is first-order on the M-TTR concentration, consistent either with a dimeric nucleus or with a nonnucleated process where each step is bimolecular and essentially irreversible. These studies suggest that amyloid formation by M-TTR under partially denaturing conditions is a downhill polymerization, in which the highest energy species is the native monomer. Our results emphasize the importance of therapeutic strategies that stabilize the TTR tetramer and may help to explain why more than eighty TTR variants are disease-associated. The differences between amyloid formation by M-TTR and other amyloidogenic peptides (such as amyloid beta-peptide and islet amyloid polypeptide) demonstrate that these polypeptides do not share a common aggregation mechanism, at least under the conditions examined thus far.

Benzothiazoles↗
PubMed2004

Metabolite-initiated protein misfolding may trigger Alzheimer's disease.

Anfinsen showed that a protein's fold is specified by its sequence. Although it is clear why mutant proteins form amyloid, it is harder to rationalize why a wild-type protein adopts a native conformation in most individuals, but it misfolds in a minority of others, in what should be a common extracellular environment. This discrepancy suggests that another event likely triggers misfolding in sporadic amyloid disease. One possibility is that an abnormal metabolite, generated only in some individuals, covalently modifies the protein or peptide and causes it to misfold, but evidence for this is sparse. Candidate metabolites are suggested by the recently appreciated links between Alzheimer's disease (AD) and atherosclerosis, known chronic inflammatory metabolites, and the newly discovered generation of ozone during inflammation. Here we report detection of cholesterol ozonolysis products in human brains. These products and a related, lipid-derived aldehyde covalently modify Abeta, dramatically accelerating its amyloidogenesis in vitro, providing a possible chemical link between hypercholesterolemia, inflammation, atherosclerosis, and sporadic AD.

Alzheimer Disease↗
PubMed2003

ALS mutants of human superoxide dismutase form fibrous aggregates via framework destabilization.

Many point mutations in human Cu,Zn superoxide dismutase (SOD) cause familial amyotrophic lateral sclerosis (FALS), a fatal neurodegenerative disorder in heterozygotes. Here we show that these mutations cluster in protein regions influencing architectural integrity. Furthermore, crystal structures of SOD wild-type and FALS mutant H43R proteins uncover resulting local framework defects. Characterizations of beta-barrel (H43R) and dimer interface (A4V) FALS mutants reveal reduced stability and drastically increased aggregation propensity. Moreover, electron and atomic force microscopy indicate that these defects promote the formation of filamentous aggregates. The filaments resemble those seen in neurons of FALS patients and bind both Congo red and thioflavin T, suggesting the presence of amyloid-like, stacked beta-sheet interactions. These results support free-cysteine-independent aggregation of FALS mutant SOD as an integral part of FALS pathology. They furthermore provide a molecular basis for the single FALS disease phenotype resulting from mutations of diverse side-chains throughout the protein: many FALS mutations reduce structural integrity, lowering the energy barrier for fibrous aggregation.

Amyotrophic Lateral Sclerosis↗
PubMed2003

Adsorption of beta-hairpin peptides on the surface of water: a neutron reflection study.

Neutron reflectivity has been used to determine the thickness and surface coverage of monolayers of two 14-residue beta-hairpin peptides adsorbed at the air/water interface. The peptides differed only in that one was labeled with a fluorophore, while the other was not. The neutron reflection measurements were mainly made in null reflecting water, NRW, containing 8.1% D(2)O. Under this isotopic contrast the water is invisible to neutrons and the specular signal was then only from the peptide layer. At the highest concentration of ca. 4 microg/mL studied, the area per peptide molecule (A) was found to be 230 +/- 10 and 210 +/- 10 A(2) for the peptides with and without a BODIPY-based fluorophore, respectively. The thickness of the peptide layers was about 10 A for a Gaussian distribution. With decreasing bulk peptide concentration, both surface excess and layer thickness showed a steady trend of decrease. While the neutron results clearly indicate structural changes within the peptide monolayers with increasing bulk concentration, the outstanding structural feature is the formation of rather uniform peptide layers, consistent with the structural characteristics typical of beta-strand peptide conformations. These structural features are well supported by the parallel measurements of the adsorbed layers in D(2)O. With this isotopic contrast the neutron reflectivity provides an estimate about the extent of immersion of the peptide layers into water. The results strongly suggest that the 14-mer peptide monolayers were fully afloat on the surface of water, with only the carboxy groups on Glu residues hydrated.

Adsorption↗
PubMed2003

Prevention of transthyretin amyloid disease by changing protein misfolding energetics.

Genetic evidence suggests that inhibition of amyloid fibril formation by small molecules should be effective against amyloid diseases. Known amyloid inhibitors appear to function by shifting the aggregation equilibrium away from the amyloid state. Here, we describe a series of transthyretin amyloidosis inhibitors that functioned by increasing the kinetic barrier associated with misfolding, preventing amyloidogenesis by stabilizing the native state. The trans-suppressor mutation, threonine 119 --> methionine 119, which is known to ameliorate familial amyloid disease, also functioned through kinetic stabilization, implying that this small-molecule strategy should be effective in treating amyloid diseases.

Amyloidosis↗