Shadoo binds lipid membranes and undergoes aggregation and fibrillization

Li, Qiaojing; Chevalier, Christophe; Henry, Céline; Richard, Charles-Adrien; Moudjou, Mohammed; Vidić, Jasmina

doi:10.1016/j.bbrc.2013.07.104

Cited by 11 publications

(18 citation statements)

References 18 publications

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“…This spontaneous conformational switch additionally points out that the C-terminal part of PB1-F2 is initially deprived of any secondary structure at these pH values. Indeed, whereas most ␣-helical proteins such as ␤ 2 -microglobulin, lysozyme, or prion protein need some chemical or thermal treatment to convert into amyloid structures in vitro (39 -41), natively disordered proteins as A␤, Shadoo, ␣-synuclain may switch to amyloid-like forms without recourse to denaturation treatment (42)(43)(44)(45).…”

Section: Discussionmentioning

confidence: 99%

Amyloid Assemblies of Influenza A Virus PB1-F2 Protein Damage Membrane and Induce Cytotoxicity

Vidić

Richard

Péchoux

et al. 2016

Journal of Biological Chemistry

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PB1-F2 is a small accessory protein encoded by an alternative open reading frame in PB1 segments of most influenza A virus. PB1-F2 is involved in virulence by inducing mitochondria-mediated immune cells apoptosis, increasing inflammation, and enhancing predisposition to secondary bacterial infections. Using biophysical approaches we characterized membrane disruptive activity of the full-length PB1-F2 (90 amino acids), its N-terminal domain (52 amino acids), expressed by currently circulating H1N1 viruses, and its C-terminal domain (38 amino acids). Both full-length and N-terminal domain of PB1-F2 are soluble at pH values <6, whereas the C-terminal fragment was found soluble only at pH < 3. All three peptides are intrinsically disordered. At pH > 7, the C-terminal part of PB1-F2 spontaneously switches to amyloid oligomers, whereas full-length and the N-terminal domain of PB1-F2 aggregate to amorphous structures. When incubated with anionic liposomes at pH 5, full-length and the C-terminal part of PB1-F2 assemble into amyloid structures and disrupt membrane at nanomolar concentrations. PB1-F2 and its C-terminal exhibit no significant antimicrobial activity. When added in the culture medium of mammalian cells, PB1-F2 amorphous aggregates show no cytotoxicity, whereas PB1-F2 pre-assembled into amyloid oligomers or fragmented nanoscaled fibrils was highly cytotoxic. Furthermore, the formation of PB1-F2 amyloid oligomers in infected cells was directly reflected by membrane disruption and cell death as observed in U937 and A549 cells. Altogether our results demonstrate that membrane-lytic activity of PB1-F2 is closely linked to supramolecular organization of the protein.

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Section: Discussionmentioning

confidence: 99%

Amyloid Assemblies of Influenza A Virus PB1-F2 Protein Damage Membrane and Induce Cytotoxicity

Vidić

Richard

Péchoux

et al. 2016

Journal of Biological Chemistry

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“…This clone was transformed into E. coli SoluBL21 bacterial strain, to express Shadoo protein with a N-terminal hexahistidine tag, (HHHHHHHHHHSSGHIDDDDKHMKGGRGGARGSARGVRGGARGASRVRVRP APRYGSSLRVAAAGAAAGAAAGVAAGLATGSGWRRTSGPGELGLEDDENGAMGGNGTDRGVYSYWAWTSG) as explained previously 11 . The plasmid can be requested from the authors.…”

Section: Generation Of Plasmid and Shadoo Expressionmentioning

confidence: 99%

“…Note: The gene encoding murine Shadoo protein (Shadoo ), was subcloned into the expression vector pET-28 11 . This clone was transformed into E. coli SoluBL21 bacterial strain, to express Shadoo protein with a N-terminal hexahistidine tag, (HHHHHHHHHHSSGHIDDDDKHMKGGRGGARGSARGVRGGARGASRVRVRP APRYGSSLRVAAAGAAAGAAAGVAAGLATGSGWRRTSGPGELGLEDDENGAMGGNGTDRGVYSYWAWTSG) as explained previously 11 .…”

Section: Generation Of Plasmid and Shadoo Expressionmentioning

confidence: 99%

“…As the N-terminal part of prion protein, Shadoo is highly positively charged and appears to be a natively unstructured protein 11,12 . Shadoo seems to be functionally related to prion disorders since it can directly bind prion protein.…”

Section: Introductionmentioning

confidence: 99%

“…In contrast, similar to prion protein, Shadoo can adopt a β-sheet-enriched structure and self-associate into amyloid fibril structures. It was shown that Shadoo can fibrillate under native conditions or upon binding negatively charged membranes 10,11 . Conversion of Shadoo into amyloid-like fibers may be associated with pathologies.…”

Section: Introductionmentioning

confidence: 99%

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Purification and Refolding to Amyloid Fibrils of (His)<sub>6</sub>-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in<em> E. coli</em>

Richard

Moudjou

et al. 2015

JoVE

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The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His) 6 -tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni 2+ -charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner. Video LinkThe video component of this article can be found at

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Lipid-associated aggregate formation of superoxide dismutase-1 is initiated by membrane-targeting loops

Chng¹,

Strange

2014

Proteins

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Copper-Zinc superoxide dismutase 1 (SOD1) is a homodimeric enzyme that protects cells from oxidative damage. Hereditary and sporadic amyotrophic lateral sclerosis may be linked to SOD1 when the enzyme is destabilized through mutation or environmental stress. The cytotoxicity of demetallated or apo-SOD1 aggregates may be due to their ability to cause defects within cell membranes by co-aggregating with phospholipids. SOD1 monomers may associate with the inner cell membrane to receive copper ions from membrane-bound copper chaperones. But how apo-SOD1 interacts with lipids is unclear. We have used atomistic molecular dynamics simulations to reveal that flexible electrostatic and zinc-binding loops in apo-SOD1 dimers play a critical role in the binding of 1-octanol clusters and phospholipid bilayer, without any significant unfolding of the protein. The apo-SOD1 monomer also associates with phospholipid bilayer via its zinc-binding loop rather than its exposed hydrophobic dimerization interface. Our observed orientation of the monomer on the bilayer would facilitate its association with a membrane-bound copper chaperone. The orientation also suggests how membrane-bound monomers could act as seeds for membrane-associated SOD1 aggregation.

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Shadoo binds lipid membranes and undergoes aggregation and fibrillization

Cited by 11 publications

References 18 publications

Amyloid Assemblies of Influenza A Virus PB1-F2 Protein Damage Membrane and Induce Cytotoxicity

Amyloid Assemblies of Influenza A Virus PB1-F2 Protein Damage Membrane and Induce Cytotoxicity

Purification and Refolding to Amyloid Fibrils of (His)<sub>6</sub>-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in<em> E. coli</em>

Lipid-associated aggregate formation of superoxide dismutase-1 is initiated by membrane-targeting loops

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