The intrinsic amyloidogenic propensity of cofilin-1 is aggravated by Cys-80 oxidation: A possible link with neurodegenerative diseases

Kaushik, Vibha; Brünnert, Daniela; Hanschmann, Eva-Maria; Sharma, Phulwanti Kumari; Anand, Bibin G.; Kar, Karunakar; Kateriya, Suneel; Goyal, Pankaj

doi:10.1016/j.bbrc.2021.07.013

Cited by 6 publications

(7 citation statements)

References 32 publications

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“…Moreover, the in vitro experiments suggested that cofilin-2 gets aggregated to form amyloid-like structures at a concentration of 25 µM, while cofilin-1 was observed to form similar structures at ~3 fold higher concentrations in vitro. Cofilin-2 formed longer amyloid fibrils compared to the shorter fibrils of cofilin-1 [20].…”

Section: Discussionmentioning

confidence: 86%

“…As described previously [20], the human cofilin-2 gene was cloned in the pET 15b plasmid using specific oligonucleotides (Forward: CATATGgcttctggagttacag; Reverse:…”

Section: Cloning Of Human Cofilin-2 Gene Expression and Purification Of The Proteinmentioning

confidence: 99%

“…The aggregation reaction was initiated by incubating 25 μM and 50 μM of the purified cofilin-2 at 37 ˚C, shaking at 1200 rpm for 24 h on a thermomixer. Further, the incubated cofilin-2 samples were assessed by Native-PAGE to confirm the formation of higher-order entities after the aggregation process, as described previously [20]. Briefly, the samples were loaded onto a 12% Tris-HCl polyacrylamide gel and electrophoresed using the mini-PROTEIN II electrophoresis system (Bio-Rad, USA) at 25 Amp and 4 ˚C throughout the process.…”

Section: Native (Non-denaturing) Polyacrylamide Gel Electrophoresis (Page)mentioning

confidence: 99%

“…Our recent study shows that Cys-80 in cofilin-1 is critical for its stability [20]. To analyze the effect of cysteine oxidation on cofilin-2 stability, we replaced the cysteine residues with aspartic acid (C to D mutation); this mimicked the formation of sulfonic acid, using Aggrescan3D 2.0.…”

Section: Both the Cysteine Residues (Cys-39 And Cys-80) In Cofilin-2 Are Critical For Its Stabilitymentioning

confidence: 99%

“…Cofilin-1 oligomerization increases during platelet activation with thrombin and is negatively regulated via phosphorylation [19]. Our recent finding revealed that cofilin-1 possesses an intrinsic propensity to form higher-order amyloidogenic fibrillar structures, and this amyloidogenesis is further aggravated by Cys-80 oxidation [20]. It has been divulged that the phosphorylated form of cofilin-2, under oxidative distress, forms fibrillar aggregates, which involves Cys-39 [4].…”

Section: Introductionmentioning

confidence: 99%

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Computational and biochemical analyses reveal that cofilin-2 self assembles into amyloid-like structures and promotes the aggregation of other proteinaceous species: Pathogenic relevance to myopathies

Kaushik

Hanschmann

Bruennert

et al. 2021

Preprint

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Cofilin-2 is a member of the ADF/cofilin family, expressed extensively in adult muscle cells and involved in muscle maintenance and regeneration. Phosphorylated cofilin-2 is found in pre-fibrillar aggregates formed during idiopathic dilated cardiomyopathy. A recent study shows that phosphorylated cofilin-2, under oxidative distress, forms fibrillar aggregates. However, it remains unknown if cofilin-2 holds an innate propensity to form amyloid-like structures. In the present study, we employed various computational and biochemical techniques to explore the amyloid-forming potential of cofilin-2. We report that cofilin-2 possesses aggregation-prone regions (APRs), and these APRs get exposed to the surface, become solvent-accessible, and are involved in the intermolecular interactions during dimerization, an early stage of aggregation. Furthermore, the cofilin-2 amyloids, formed under physiological conditions, are capable of cross-seeding other monomeric globular proteins and amino acids, thus promoting their aggregation. We further show that Cys-39 and Cys-80 are critical in maintaining the thermodynamic stability of cofilin-2. The destabilizing effect of oxidation at Cys-39 but not that at Cys-80 is mitigated by Ser-3 phosphorylation. Cysteine oxidation leads to partial unfolding and loss of structure, suggesting that cysteine oxidation further induces early events of cofilin-2 aggregation. Overall, our results pose a possibility that cofilin-2 amyloidogenesis might be involved in the pathophysiology of diseases, such as myopathies. We propose that the exposure of APRs to the surface could provide mechanistic insight into the higher-order aggregation and amyloidogenesis of cofilin-2. Moreover, the cross-seeding activity of cofilin-2 amyloids hints towards its involvement in the hetero-aggregation in various amyloid-linked diseases.

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Section: Discussionmentioning

confidence: 86%

“…As described previously [20], the human cofilin-2 gene was cloned in the pET 15b plasmid using specific oligonucleotides (Forward: CATATGgcttctggagttacag; Reverse:…”

Section: Cloning Of Human Cofilin-2 Gene Expression and Purification Of The Proteinmentioning

confidence: 99%

Section: Native (Non-denaturing) Polyacrylamide Gel Electrophoresis (Page)mentioning

confidence: 99%

Section: Both the Cysteine Residues (Cys-39 And Cys-80) In Cofilin-2 Are Critical For Its Stabilitymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Computational and biochemical analyses reveal that cofilin-2 self assembles into amyloid-like structures and promotes the aggregation of other proteinaceous species: Pathogenic relevance to myopathies

Kaushik

Hanschmann

Bruennert

et al. 2021

Preprint

Self Cite

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Protein–protein interactions regulating α-synuclein pathology

Wang,

Dai,

Chen

et al. 2024

Trends in Neurosciences

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Ellman’s Assay on the Surface: Thiol Quantification of Human Cofilin-1 Protein through Surface Plasmon Resonance

Souza,

Monteiro,

Guimarães

et al. 2024

Langmuir

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Oxidative stress on cysteine (Cys)-containing proteins has been associated with physiological disorders, as suggested for the human cofilin-1 (CFL-1) protein, in which the oxidized residues are likely implicated in the aggregation process of αsynuclein, leading to severe neuronal injuries. Considering the relevance of the oxidation state of cysteine, quantification of thiols may offer a guide for the development of effective therapies. This work presents, for the very first time, thiol quantification within CFL-1 in solution and on the surface following classic and adapted versions of Ellman's assay. The 1:1 stoichiometric Ellman's reaction occurs between 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), and the free thiol of the cysteine residue, producing two 2nitro-5-thiobenzoate (TNB 2− ) ions, one of which is released into the medium. While in solution, the thiol concentration was determined by the absorbance of the released TNB 2− , on the surface, the mass of the attached TNB 2− ion to the protein allowed the quantification by means of the multiparametric surface plasmon resonance (MP-SPR) technique. The SPR angle change after the interaction of DTNB with immobilized CFL-1 gave a surface coverage of 26.5 pmol cm −2 for the TNB 2− ions (Γ TNB2− ). The ratio of this value to the surface coverage of CFL-1, Γ CFL-1 = 6.5 ± 0.6 pmol cm −2 (also determined by MP-SPR), gave 4.1 as expected for this protein, i.e., CFL-1 contains four Cys residues in its native form (reduced state). A control experiment with adsorbed oxidized protein showed no SPR angle change, thus proving the reliability of adapting Ellman's assay to the surface using the MP-SPR technique. The results presented in this work provide evidence of the heterogenization of Ellman's assay, offering a novel perspective for studying thiol-containing species within proteins. This may be particularly useful to ensure further studies on drug-like molecules that can be carried out with validated oxidized or reduced CFL-1 or other analogous systems.

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The intrinsic amyloidogenic propensity of cofilin-1 is aggravated by Cys-80 oxidation: A possible link with neurodegenerative diseases

Cited by 6 publications

References 32 publications

Computational and biochemical analyses reveal that cofilin-2 self assembles into amyloid-like structures and promotes the aggregation of other proteinaceous species: Pathogenic relevance to myopathies

Computational and biochemical analyses reveal that cofilin-2 self assembles into amyloid-like structures and promotes the aggregation of other proteinaceous species: Pathogenic relevance to myopathies

Protein–protein interactions regulating α-synuclein pathology

Ellman’s Assay on the Surface: Thiol Quantification of Human Cofilin-1 Protein through Surface Plasmon Resonance

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