Agitation of amyloid proteins to speed aggregation measured by ThT fluorescence: A call for standardization

Batzli, Kiersten M.; Love, Brian J.

doi:10.1016/j.msec.2014.09.015

Cited by 39 publications

(29 citation statements)

References 47 publications

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“…3B). Although ThT data suggested that mature fibrils exist by this point in aggregation, AFM was performed under quiescent conditions, which delays fibrillation (46). When DLPC SLBs were treated with A␤, a large number of globular aggregates were present on the bilayer after 12 h. Aggregates persisted after a 24-h incubation, although the size of individual particles shrank.…”

Section: Resultsmentioning

confidence: 99%

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Reduced Lipid Bilayer Thickness Regulates the Aggregation and Cytotoxicity of Amyloid-β

Korshavn

Satriano

Lin

et al. 2017

Journal of Biological Chemistry

157

158

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Edited by Paul E. FraserThe aggregation of amyloid-␤ (A␤) on lipid bilayers has been implicated as a mechanism by which A␤ exerts its toxicity in Alzheimer's disease (AD). Lipid bilayer thinning has been observed during both oxidative stress and protein aggregation in AD, but whether these pathological modifications of the bilayer correlate with A␤ misfolding is unclear. Here, we studied peptide-lipid interactions in synthetic bilayers of the shortchain lipid dilauroyl phosphatidylcholine (DLPC) as a simplified model for diseased bilayers to determine their impact on A␤ aggregate, protofibril, and fibril formation. A␤ aggregation and fibril formation in membranes composed of dioleoyl phosphatidylcholine (DOPC) or 1-palmitoyl-2-oleoyl phosphatidylcholine mimicking normal bilayers served as controls. Differences in aggregate formation and stability were monitored by a combination of thioflavin-T fluorescence, circular dichroism, atomic force microscopy, transmission electron microscopy, and NMR. Despite the ability of all three lipid bilayers to catalyze aggregation, DLPC accelerates aggregation at much lower concentrations and prevents the fibrillation of A␤ at low micromolar concentrations. DLPC stabilized globular, membrane-associated oligomers, which could disrupt the bilayer integrity. DLPC bilayers also remodeled preformed amyloid fibrils into a pseudo-unfolded, molten globule state, which resembled on-pathway, protofibrillar aggregates. Whereas the stabilized, membrane-associated oligomers were found to be nontoxic, the remodeled species displayed toxicity similar to that of conventionally prepared aggregates. These results provide mechanistic insights into the roles that pathologically thin bilayers may play in A␤ aggregation on neuronal bilayers, and pathological lipid oxidation may contribute to A␤ misfolding.

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Section: Resultsmentioning

confidence: 99%

“…Normalized curves were then individually fit to Equations 1 and 2 to calculate the individual lag phase (t lag ) for each curve (46). These values were then averaged across three separate trials.…”

Section: Methodsmentioning

confidence: 99%

Reduced Lipid Bilayer Thickness Regulates the Aggregation and Cytotoxicity of Amyloid-β

Korshavn

Satriano

Lin

et al. 2017

Journal of Biological Chemistry

157

158

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“…Despite the frequent use of agitation little is known about the shear forces that are produced and the effects on different steps in the aggregation reaction ( figure 4). In addition, as stirring/shaking is often different in different studies, it is challenging to undertake a comparative analysis on systems studied in different research groups [241].…”

Section: Agitationmentioning

confidence: 99%

Factors affecting the physical stability (aggregation) of peptide therapeutics

Zapadka

Becher

Santos³

et al. 2017

Interface Focus.

265

207

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The number of biological therapeutic agents in the clinic and development pipeline has increased dramatically over the last decade and the number will undoubtedly continue to increase in the coming years. Despite this fact, there are considerable challenges in the development, production and formulation of such biologics particularly with respect to their physical stabilities. There are many cases where self-association to form either amorphous aggregates or highly structured fibrillar species limits their use. Here, we review the numerous factors that influence the physical stability of peptides including both intrinsic and external factors, wherever possible illustrating these with examples that are of therapeutic interest. The effects of sequence, concentration, pH, net charge, excipients, chemical degradation and modification, surfaces and interfaces, and impurities are all discussed. In addition, the effects of physical parameters such as pressure, temperature, agitation and lyophilization are described. We provide an overview of the structures of aggregates formed, as well as our current knowledge of the mechanisms for their formation.

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“…Samples without seeds were fitted to the proposed standard model for ThT assays by Batzli and Love, 41 and the proposed kvalues were used as rate constants for fibril elongation. Lag time was identified as the time point value of the intersection between linear regression lines of the lag time phase and the fibril growth phase.…”

Section: Data Processing and Fitting To Modelmentioning

confidence: 99%

Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin

Hjorth

Norrman

Wahlund

et al. 2016

Journal of Pharmaceutical Sciences

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A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation.

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Agitation of amyloid proteins to speed aggregation measured by ThT fluorescence: A call for standardization

Cited by 39 publications

References 47 publications

Reduced Lipid Bilayer Thickness Regulates the Aggregation and Cytotoxicity of Amyloid-β

Reduced Lipid Bilayer Thickness Regulates the Aggregation and Cytotoxicity of Amyloid-β

Factors affecting the physical stability (aggregation) of peptide therapeutics

Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin

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