Investigation of a Peptide Responsible for Amyloid Fibril Formation of β2-Microglobulin byAchromobacter Protease I

Kozhukh, Genadiy; Hagihara, Yoshihisa; Kawakami, Toru; Hara, Kazuhiro; Naiki, Hironobu; Goto, Yuji

doi:10.1074/jbc.m108753200

Cited by 119 publications

(161 citation statements)

References 33 publications

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“…Two peptides (residues 21-40, the so-called K3 peptide, and residues 59-71), which overlap with the regions that are strongly exchange broadened in the pH 3.6 intermediate, were reported to self-associate in Vitro. 22,23 The two -strands (residues 21-28 and 33-40) observed in amyloid fibrils of the K3 peptide 24 match the two minima in the H/D exchange profile (Figure 2), suggesting that amyloid fibrils of full-length b2m also contain two -strands in this region.We demonstrated that signals of partially unfolded intermediate ensembles broadened due to conformational exchange can be structurally characterized using direct carbon-detected NMR experiments. Comparison between the structural and dynamic properties of the amyloid intermediate of b2m with H/D exchange measurements on amyloid fibrils revealed a close relationship between the conformational properties of the metastable partially unfolded precursor and the -sheet-rich insoluble aggregates of a diseaserelevant protein that assumes a rigid 3D fold in its native state.…”

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confidence: 68%

Molten Globule Precursor States Are Conformationally Correlated to Amyloid Fibrils of Human β-2-Microglobulin

2010

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Misfolding intermediates play a key role in defining aberrant protein aggregation and amyloid formation in more than 15 different human diseases. However, their experimental characterization is challenging due to the transient nature and conformational heterogeneity of the involved states. Here, we demonstrate that direct carbon-detected NMR experiments allow observation, assignment, and structural analysis of molten globule amyloid intermediates that are severely broadened by conformational exchange. The method is used to characterize the structure and dynamics of partially unfolded intermediates of the 99-residue protein -2-microglobulin, which is the major component of insoluble aggregates occurring in dialysis-related amyloidosis. Comparison of the conformational properties of the molten globule-like intermediates with levels of deuterium incorporation into amyloid fibrils of -2-microglobulin revealed a close relationship between the conformational properties of the metastable intermediates and the -sheet-rich insoluble aggregates of -2-microglobulin.Misfolding intermediates play a key role in defining aberrant protein aggregation and amyloid formation in more than 15 different human diseases. 1,2 However, the experimental characterization of the conformation of amyloid intermediates is challenging due to their transient nature and conformational heterogeneity. 3,4 This severely limits our knowledge about the relation of the conformation of the precursor states to the structure of amyloid fibrils.Dialysis-related amyloidosis is a protein misfolding disease resulting from deposition of amyloid aggregates in skeletal tissue that contain fibrils of the 99-residue-long protein -2-microglobulin (b2m). 5 Amyloid formation of b2m is strongly enhanced in conditions that destabilize its globular structure. 6 This can be achieved in Vitro by acid denaturation, with two distinct intermediate states being formed under acidic conditions. The highest population of the partially unfolded intermediate occurs at pH 3.6, where also the rate of fibril formation reaches a maximum. 7 However, long and straight amyloid fibrils resembling those extracted from patients are formed from a precursor state formed at pH 2.5. 8,9 Here, we compare the conformational properties of these two partially unfolded intermediates with single-residue resolution and demonstrate that a close relationship exists between the structure and dynamics of the amyloid precursor species and the morphology of mature fibrils of b2m.At pH 2.5 b2m is highly unfolded, the majority of resonances are observed in 1 H-15 N HSQC spectra (Supporting Information, Figure 1) and can be assigned to individual residues. 10 In contrast, only about 40-60 resolved peaks on top of a broad hump of unresolved signal intensity (visible at lower contour levels) could be observed at pH 3.6 ( Figure 1a). 11 To obtain sequence-specific information about the pH 3.6 intermediate, we tested direct 13 C detection. 12-14 Direct carbon detection was previously successfully used fo...

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confidence: 68%

Molten Globule Precursor States Are Conformationally Correlated to Amyloid Fibrils of Human β-2-Microglobulin

2010

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“…We found that a 22-residue K3 peptide, Ser 20 -Lys 41 ( Fig. 1), obtained by digestion of ␤ 2 m with Acromobacter protease I, forms amyloid fibrils (30). Recently, we further identified an 11-residue peptide, Asn 21 -His 31 , in the K3 region that also forms amyloid fibrils (42).…”

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confidence: 95%

“…Recombinant ␤ 2 m and K3 Peptide-Recombinant human ␤ 2 m was expressed in the methylotrophic yeast, Pichia pastoris, and purified as described (29,30). Three fractions with 6 (Glu-Ala-Glu-Ala-Tyr-Val-), 4 (Glu-Ala-Tyr-Val-), and 1 (Val-) additional amino acid residues added to the N-terminal (Leu) of intact ␤ 2 m were obtained.…”

Section: Methodsmentioning

confidence: 99%

Optimum Amyloid Fibril Formation of a Peptide Fragment Suggests the Amyloidogenic Preference of β2-Microglobulin under Physiological Conditions

Ohhashi

Hara

Naiki

et al. 2004

Journal of Biological Chemistry

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␤ 2 -Microglobulin (␤ 2 m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Although full-length ␤ 2 m readily forms amyloid fibrils in vitro by seed-dependent extension with a maximum at pH 2.5, fibril formation under physiological conditions as detected in patients has been difficult to reproduce. A 22-residue K3 peptide of ␤ 2 m, Ser 20 -Lys 41 , obtained by digestion with Acromobacter protease I, forms amyloid fibrils without seeding. To obtain further insight into the mechanism of fibril formation, we studied the pH dependence of fibril formation of the K3 peptide and its morphology using a ThT fluorescence assay and electron microscopy, respectively. K3 peptide formed amyloid fibrils over a wide range of pH values with an optimum around pH 7 and contrasted with the pH profile of the seed-dependent extension reaction of full-length ␤ 2 m. This suggests that once the rigid native-fold of ␤ 2 m is unfolded and additional factors triggering the nucleation process are provided, full-length ␤ 2 m discloses an intrinsic potential to form amyloid fibrils at neutral pH. The fibril formation was strongly promoted by dimerization of K3 through Cys 25 . The morphology of the fibrils varied depending on the fibril formation conditions and the presence or absence of a disulfide bond. Various fibrils had the potential to seed fibril formation of full-length ␤ 2 m accompanied with a characteristic lag phase, suggesting that the internal structures are similar.Amyloid fibrils are recognized as being associated with the pathology of more than 20 serious human diseases and the responsible peptides or proteins specific to these diseases have been identified (1-5). These fibrils are characterized by a cross-␤ structure where ␤-strands are perpendicularly oriented to the axis of the polymeric fibril (6 -8). Moreover, various proteins and peptides that are not related to diseases can also form amyloid-like fibers, implying that formation of amyloid fibrils is a general property of polypeptides (7, 9 -14). Clarifying the mechanism of amyloid fibril formation is essential not only for understanding the pathogenesis of amyloidosis but also for improving our understanding of the mechanism of protein folding.Dialysis-related amyloidosis is a common and serious complication among patients on long term hemodialysis (15, 16), in which ␤ 2 -microglobulin (␤ 2 m) 1 forms amyloid fibrils. Native ␤ 2 m, made of 99 amino acid residues, corresponds to a typical immunoglobulin domain ( Fig. 1) and is a component of the type I major histocompatibility antigen (17)(18)(19). Although the increase in ␤ 2 m concentration in blood over a long period is the most critical risk factor causing amyloidosis, the molecular details remain unknown. Recently ␤ 2 m, because of its relatively small size, which makes it suitable for physicochemical studies, has been used as a target for extensive studies addressing the mechanism of amyloid fibril formation in the context of protein conformation (20 -28). We have st...

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“…K3 was obtained by digestion of ␤2-m with lysyl endopeptidase (Achromobacter protease I) as reported previously (16).…”

Section: Methodsmentioning

confidence: 99%

Flow-induced Alignment of Amyloid Protofilaments Revealed by Linear Dichroism

Adachi

Yamaguchi

Yagi

et al. 2007

Journal of Biological Chemistry

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Amyloid fibrils underlying various serious amyloidoses including Alzheimer and prion diseases form characteristic deposits in which linear fibrils with an unbranched and rigid morphology associate laterally or radially, e.g. radial senile amyloid plaques of amyloid ␤. To clarify the formation of these high order amyloid deposits, studying the rheology is important. A 22-residue K3 peptide fragment of ␤ 2 -microglobulin, a protein responsible for dialysis-related amyloidosis, forms long and homogeneous protofilament-like fibrils in 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl (pH ϳ2). Here, using circular dichroism and linear dichroism, we observed the flow-induced alignment of fibrils. Analysis of far-and near-UV linear dichroism spectra suggested that both the net -* transition moment of the backbone carbonyl group and L b transition moment of the Tyr 26 side chain are oriented in parallel to the fibril axis, revealing the structural details of amyloid protofilaments. Moreover, the intensities of flow-induced circular dichroism or linear dichroism signals depended critically on the length and type of fibrils, suggesting that they are useful for detecting and characterizing amyloid fibrils.Although the structural features of amyloid fibrils are of urgent importance (1-3), the high molecular weight and noncrystalline assembly preclude the use of conventional methods, such as x-ray crystallography or multidimensional NMR, to acquire high resolution structural images. Recently, it has been found that amyloid fibrils are, in general, amenable to the magic angle-spinning solid-state nuclear magnetic resonance methods, providing a general view of the atomic level structure in which ␤-strands are stacked in a parallel and registered manner (4 -6). A structure revealed by x-ray diffraction of a crystalline short amyloidogenic peptide was consistent with this view (7).More recently, the mechanical characterization of individual fibrils by atomic force microscopy revealed that the insulin fibrils have a strength and stiffness comparable with that of steel and silk, respectively (8). Among various structural features, although the long and rigid morphology of amyloid fibrils might be responsible for their biological consequences, rheological details remain unknown.␤ 2 -Microglobulin (␤2-m), 4 a typical immunoglobulin domain made of 99 residues, is a target of extensive study because of its clinical importance and suitable size for examining the relationship between protein folding and amyloid formation (9 -14). Dialysis-related amyloidosis is a common and serious complication among patients on long term hemodialysis, in which amyloid fibrils of ␤2-m deposit in the synovia of the carpal tunnel (9, 15). A 22-residue K3 peptide, corresponding to Ser 20 -Lys 41 of ␤2-m forms protofilament-like fibrils in various solvents including a low concentration of 2,2,2-trifluoroethanol (16 -19). Although the efficient fibrillation of intact ␤2-m requires seeding, K3 forms fibrils spontaneously. Furthermore, K3 formed two types of fibr...

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Investigation of a Peptide Responsible for Amyloid Fibril Formation of β2-Microglobulin byAchromobacter Protease I

Cited by 119 publications

References 33 publications

Molten Globule Precursor States Are Conformationally Correlated to Amyloid Fibrils of Human β-2-Microglobulin

Molten Globule Precursor States Are Conformationally Correlated to Amyloid Fibrils of Human β-2-Microglobulin

Optimum Amyloid Fibril Formation of a Peptide Fragment Suggests the Amyloidogenic Preference of β2-Microglobulin under Physiological Conditions

Flow-induced Alignment of Amyloid Protofilaments Revealed by Linear Dichroism

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