Refolding and Polymerization Pathways of Neuroserpin

Takehara, Shoichiro; Zhang, Juan; Yang, Xiao-Yan; Takahashi, Nobuyuki; Mikami, Bunzo; Onda, Maki

doi:10.1016/j.jmb.2010.07.047

Cited by 20 publications

(30 citation statements)

References 29 publications

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“…The stress-inducible mutant antithrombin London is also able to polymerize by forming disulfide intermolecular linkages intracellularly. This polymerization might be explained by the existence of intermediate folding states in the endoplasmic reticulum during protein synthesis, which can follow a more stable conformation under these conditions, remaining accumulated inside the cells or being driven to the proteasome (41)(42)(43). Antithrombin has three disulfide bridges that should be formed early during folding.…”

Section: Discussionmentioning

confidence: 99%

“…By attaining some homology with the polymerization of neuroserpin (42) and α1-antitrypsin (13,43), both mutations could affect the previous step to achieve native conformation, in which the s1C-s4B-s5B region cannot efficiently associate with the already folded N-terminal region. The P1 mutations might slow down the last folding step, when all disulfide bridges are already formed (13,43). Thus, the mutation does not reduce secretion as much as seen with the constitutive mutants, and some protein ends up in the plasma.…”

Section: Discussionmentioning

confidence: 99%

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The Infective Polymerization of Conformationally Unstable Antithrombin Mutants May Play a Role in the Clinical Severity of Antithrombin Deficiency

Martínez‐Martínez

Navarro-Fernández

Águila

et al. 2012

Mol Med

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Mutations affecting mobile domains of antithrombin induce conformational instability resulting in protein polymerization that associates with a severe clinical phenotype, probably by an unknown gain of function. By homology with other conformational diseases, we speculated that these variants might infect wild-type (WT) monomers reducing the anticoagulant capacity. Infective polymerization of WT polymers and different P1 mutants (p.R425del, p.R425C and p.R425H) were evaluated by using native gels and radiolabeled WT monomers and functional assays. Human embryonic kidney cells expressing the Epstein-Barr nuclear antigen 1 (HEK-EBNA) cells expressing inducible (p.R425del) or two novel constitutive (p.F271S and p.M370T) conformational variants were used to evaluate intracellular and secreted antithrombin under mild stress (pH 6.5 and 39°C for 5 h). We demonstrated the conformational sensitivity of antithrombin London (p.R425del) to form polymers under mild heating. Under these conditions purified antithrombin London recruited WT monomers into growing polymers, reducing the anticoagulant activity. This process was also observed in the plasma of patients with p.R425del, p.R425C and p.R425H mutations. Under moderate stress, coexpression of WT and conformational variants in HEK-EBNA cells increased the intracellular retention of antithrombin and the formation of disulfide-linked polymers, which correlated with impaired secretion and reduction of anticoagulant activity in the medium. Therefore, mutations inducing conformational instability in antithrombin allow its polymerization with the subsequent loss of function, which under stress could sequestrate WT monomers, resulting in a new prothrombotic gain of function, particularly relevant for intracellular antithrombin. The in vitro results suggest a temporal and severe plasma antithrombin deficiency that may contribute to the development of the thrombotic event and to the clinical severity of these mutations.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The Infective Polymerization of Conformationally Unstable Antithrombin Mutants May Play a Role in the Clinical Severity of Antithrombin Deficiency

Martínez‐Martínez

Navarro-Fernández

Águila

et al. 2012

Mol Med

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“…Kim et al denatured a1AT molecules completely in 7 M urea for 3 min at 25 C, refolded in different concentrations of urea after manual mixing with a dead time of 5 s, and characterized the products by the fluorescence (Kim and Yu, 1996). Recent work on neuroserpin by Takehara et al is in good agreement with what was found for a1AT (Takehara et al, 2010). Both studies emphasized the persistence of a folding intermediate in mutated a1AT.…”

Section: Refoldingmentioning

confidence: 65%

Serpin Polymerization In Vitro

Huntington

Yamasaki

2011

Methods in Enzymology

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“…Conditions for fibril formation were generally 30 lM monomer in a buffer of 50 mM acetic acid/sodium acetate and 50 mM phosphoric acid/NaH 2 PO 4 at pH 2 or pH 5 with 0.5-1.0M NaCl at 22 C, unless otherwise indicated. Kinetic experiments at different conditions were independently replicated three to five times; the variation in the observed rate was <30%.…”

Section: Preparation and Analysis Of Histone Fibrilsmentioning

confidence: 99%

“…As illustrated in Figure 1, there is pseudo-symmetry between the interactions of the N-terminal half of H3 (blue) and the C-terminal half of H4 (red) compared to the N-terminal half of H4 (purple) and the C-terminal half of H3 (cyan). In addition to mediating appropriate oligomerization, 3D domain swapping has been implicated in the propensity of proteins to aggregate into highly ordered structures such as crystals, 21 polymers, 22 fibers, 23 and fibrils. [24][25][26] Of particular interest is the possible connection between domain swapping and formation of amyloid or amyloid-like fibrils (for review see Ref.…”

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confidence: 99%

The impact of solubility and electrostatics on fibril formation by the H3 and H4 histones

Topping

Gloss

2011

Protein Science

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The goal of this study was to examine fibril formation by the heterodimeric eukaryotic histones (H2A-H2B and H3-H4) and homodimeric archaeal histones (hMfB and hPyA1). The histone fold dimerization motif is an obligatorily domain-swapped structure comprised of two fused helix:b-loop:helix motifs. Domain swapping has been proposed as a mechanism for the evolution of protein oligomers as well as a means to form precursors in the formation of amyloid-like fibrils. Despite sharing a common fold, the eukaryotic histones of the core nucleosome and archaeal histones fold by kinetic mechanisms of differing complexity with transient population of partially folded monomeric and/or dimeric species. No relationship was apparent between fibrillation propensity and equilibrium stability or population of kinetic intermediates. Only H3 and H4, as isolated monomers and as a heterodimer, readily formed fibrils at room temperature, and this propensity correlates with the significantly lower solubility of these polypeptides. The fibrils were characterized by ThT fluorescence, FTIR, and far-UV CD spectroscopies and electron microscopy. The helical histone fold comprises the protease-resistant core of the fibrils, with little or no protease protection of the poorly structured N-terminal tails. The highly charged tails inhibit fibrillation through electrostatic repulsion. Kinetic studies indicate that H3 and H4 form a co-fibril, with simultaneous incorporation of both histones. The potential impact of H3 and H4 fibrillation on the cytotoxicity of extracellular histones and a-synuclein-mediated neurotoxicity and fibrillation is considered.

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Refolding and Polymerization Pathways of Neuroserpin

Cited by 20 publications

References 29 publications

The Infective Polymerization of Conformationally Unstable Antithrombin Mutants May Play a Role in the Clinical Severity of Antithrombin Deficiency

The Infective Polymerization of Conformationally Unstable Antithrombin Mutants May Play a Role in the Clinical Severity of Antithrombin Deficiency

Serpin Polymerization In Vitro

The impact of solubility and electrostatics on fibril formation by the H3 and H4 histones

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