Amyloid Protofilaments from the Calcium-binding Protein Equine Lysozyme: Formation of Ring and Linear Structures Depends on pH and Metal Ion Concentration

Mališauskas, Mantas; Zamotin, Vladimir; Jaß, Jana; Noppe, Wim; Dobson, Christopher M.; Morozova‐Roche, Ludmilla A.

doi:10.1016/s0022-2836(03)00551-5

Cited by 113 publications

(144 citation statements)

References 67 publications

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“…2b). Similar protofilaments were observed previously at pH 4.5 and 57°C in the presence of 10 mM CaCl 2 (18).…”

Section: Resultssupporting

confidence: 89%

“…It is interesting to note that EL can assemble into amyloid rings in a calcium-dependent manner (18). However, under the conditions of cytotoxicity assay, calcium ions are present in the culture medium, and the proportion of rings varies in both samples incubated at pH 2.0 and 4.5.…”

Section: Discussionmentioning

confidence: 99%

“…Protein concentration was determined by absorbance measurements on a Beckman UV-VIS spectrophotometer at 280 nm using an extinction coefficient, E 1% ϭ 23.5. To produce amyloid structures, EL was incubated at a 20 mg/ml (1.36 mM) concentration in 20 mM glycine (pH 2.0) or 10 mM sodium acetate (pH 4.5) buffers with 0.2% sodium azide at 57°C, as described previously (18).…”

Section: Methodsmentioning

confidence: 99%

“…Oligomers of human ␣-lactalbumin (HAM-LET) appeared to be toxic to immature and tumor cells (11). Recently, we have demonstrated that, in contrast to lysozymes and ␣-lactalbumins (16,17), EL forms morphologically very distinct linear and annular protofilaments, depending on calcium ion concentrations and pH (18). In the present study, the soluble amyloid oligomers of EL were produced under fibrillation conditions prior to development of the filamentous structures, and their toxicity was assessed in comparison with the monomeric and fibrillar species.…”

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confidence: 99%

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Does the Cytotoxic Effect of Transient Amyloid Oligomers from Common Equine Lysozyme in Vitro Imply Innate Amyloid Toxicity?

Mališauskas

Östman

Darinskas

et al. 2005

Journal of Biological Chemistry

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In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57°C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to octamer and 20-mer. Monomeric lysozyme and protofilaments are not cytotoxic, whereas the oligomers induce cell death in primary neuronal cells, primary fibroblasts, and the neuroblastoma IMR-32 cell line. Cytotoxicity was accessed by ethidium bromide staining, MTT reduction, and TUNEL assays. Primary cultures were more susceptible to the toxic effect induced by soluble amyloid oligomers than the neuroblastoma cell line. The cytotoxicity correlates with the size of oligomers; the sample incubated at pH 4.5 and containing larger oligomers, including 20-mer, appears to be more cytotoxic than the lysozyme sample kept at pH 2.0, in which only tetramers and octamers were found. Soluble amyloid oligomers may assemble into rings; however, there was no correlation between the quantity of rings in the sample and its toxicity. The cytotoxicity of transient oligomeric species of the ubiquitous protein lysozyme indicates that this is an intrinsic feature of protein amyloid aggregation, and therefore soluble amyloid oligomers can be used as a primary therapeutic target and marker of amyloid disease.The molecular basis of the pathogenicity of amyloid aggregates is a central theme in understanding the causes of a wide range of amyloid-related diseases, including Alzheimer's, Parkinson's, prion diseases, type II diabetes, and familial amyloidotic polyneuropathy (1-4). There is a striking difference between the amounts of amyloid depositions in various types of amyloid disorders. In systemic lysozyme amyloidosis, for example, the deposits can grow to kilogram quantities in the liver (5, 6). In neurodegenerative diseases, by contrast, there is no clear correlation between the amount of amyloid deposition and the clinical severity of disease. Significant cognitive impairment of Alzheimer's patients was observed without noticeable amyloid deposits in the brain, although the level of readily soluble amyloid oligomeric assemblies in the Alzheimer's brain was found to be greatly elevated (7,8).The evidence is accumulating that prefibrillar aggregates are cytotoxic both in vivo and in vitro (4,8,9), although this question is still open to debate. Moreover, aggregates of the proteins, which are not related to clinical amyloidoses, such as human ␣-lactalbumin (10), SH3 domain, or HypF-N protein, have also been found to be cytotoxic, which implies a common mechanism for cytotoxicity of misfolded proteins (11). By contrast, it has been shown that the mature amyl...

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“…2b). Similar protofilaments were observed previously at pH 4.5 and 57°C in the presence of 10 mM CaCl 2 (18).…”

Section: Resultssupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Does the Cytotoxic Effect of Transient Amyloid Oligomers from Common Equine Lysozyme in Vitro Imply Innate Amyloid Toxicity?

Mališauskas

Östman

Darinskas

et al. 2005

Journal of Biological Chemistry

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“…Typical amyloid protein deposits consist of linear fibrils 60 -130 Å wide and 1-6 microns long (5) with cross-␤ structure (6,7) and exhibiting birefringence of bound Congo red under polarized light (8). The ability to form fibrils is independent of the origin, structural class, or function of the protein or polypeptide (9 -12); in addition, non-amyloidogenic, stable globular proteins have also been observed to form fibrils on destabilization (13)(14)(15)(16)(17)(18). These observations have led to a common belief that a conformational switch from the native conformation is responsible for fibrillation (2, 19 -23).…”

mentioning

confidence: 99%

Stimulation of Insulin Fibrillation by Urea-induced Intermediates

Ahmad

Millett

Doniach

et al. 2004

Journal of Biological Chemistry

105

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Fibrillar deposits of insulin cause serious problems in implantable insulin pumps, commercial production of insulin, and for some diabetics. We performed a systematic investigation of the effect of urea-induced structural perturbations on the mechanism of fibrillation of insulin. The addition of as little as 0.5 M urea to zincbound hexameric insulin led to dissociation into dimers. Moderate concentrations of urea led to accumulation of a partially unfolded dimer state, which dissociates into an expanded, partially folded monomeric state. Very high concentrations of urea resulted in an unfolded monomer with some residual structure. The addition of even very low concentrations of urea resulted in increased fibrillation. Accelerated fibrillation correlated with population of the partially folded intermediates, which existed at up to 8 M urea, accounting for the formation of substantial amounts of fibrils under such conditions. Under monomeric conditions the addition of low concentrations of urea slowed down the rate of fibrillation, e.g. 5-fold at 0.75 M urea. The decreased fibrillation of the monomer was due to an induced non-native conformation with significantly increased ␣-helical content compared with the native conformation. The data indicate a close-knit relationship between insulin conformation and propensity to fibrillate. The correlation between fibrillation and the partially unfolded monomer indicates that the latter is a critical amyloidogenic intermediate in insulin fibrillation.

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Amyloids and Protein Aggregation—Analytical Methods

Li¹,

Rahimi²,

Sinha³

et al. 2009

Encyclopedia of Analytical Chemistry

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More than 30 diseases are associated with amyloid‐forming proteins, which undergo conformational alterations and “misfolding” under particular conditions. These proteins deposit as insoluble proteinaceous aggregates generating disease‐specific histopathologic lesions. The proteins contributing to each disease have dissimilar sequences and native structures, yet they share the commonality of forming insoluble amyloid aggregates characterized by fibrillar morphology and cross‐β structure. Presently, it is thought that the predominant agents causing cell toxicity and tissue damage are soluble, prefibrillar assemblies of these proteins. Because of the metastable nature of these prefibrillar assemblies and the noncrystalline nature of fibrillar protein aggregates, structural study of amyloid proteins is difficult. As a result, structural characterization of these proteins is typically done using combinations of low‐resolution analytical methods. Here, we present a compendium of analytical methods used to study the secondary, tertiary, and quaternary structures, and morphology of prefibrillar and fibrillar assemblies of amyloid‐forming proteins.

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Amyloid Protofilaments from the Calcium-binding Protein Equine Lysozyme: Formation of Ring and Linear Structures Depends on pH and Metal Ion Concentration

Cited by 113 publications

References 67 publications

Does the Cytotoxic Effect of Transient Amyloid Oligomers from Common Equine Lysozyme in Vitro Imply Innate Amyloid Toxicity?

Does the Cytotoxic Effect of Transient Amyloid Oligomers from Common Equine Lysozyme in Vitro Imply Innate Amyloid Toxicity?

Stimulation of Insulin Fibrillation by Urea-induced Intermediates

Amyloids and Protein Aggregation—Analytical Methods

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